Human adipocyte cell populations and methods for identifying modulators of same

ABSTRACT

The invention features methods of obtaining high-yield, essentially pure human predipocyte cultures. Cultures obtained according to the instant methodology are also featured as are methods of identifying adipogenic modulatory agents, e.g., high-throughput screening assays.

RELATED APPLICATIONS

This application claims the benefit of prior-filed U.S. provisionalpatent application Ser. No. 60/377,500, entitled “Human Adipocyte CellPopulations and Methods for Identifying Modulators”, filed May 1, 2002(pending).

GOVERNMENT RIGHTS

This invention was made at least in part with government support undergrant no. 1-R43-DK-54588-1 awarded by the National Institutes of Health.The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Obesity is a well-established risk factor for many common diseases,including diabetes, coronary heart disease, hypertension,osteoarthritis, gallbladder disease, and colon, endometrial, and breastcancer. Visceral obesity is a particularly important risk factor forthese diseases. Over one in three Americans are overweight, with asubstantial economic effect. Annual direct healthcare costs of diseaseattributable to obesity in the U.S. were estimated at approximately 52billion dollars in 1995, or 5.7% of our national health expenditure thatyear. Most of the direct costs (63%) were from type 2 diabetes. Thepotential market for anti-obesity treatments exceeds $33 billion inNorth America annually.

There is no cure for obesity. Current methods for managing obesityinclude appetite suppressant drugs, drugs that block intestinalabsorption of nutrients, diets, surgery, and behavioral approaches.Results of these treatments have been disappointing: only a smallpercentage of weight is lost and this is typically regained. Existingdrug and dietary treatments for obesity are only modestly effective.Current treatments for obesity focus on dieting, surgery, and drugs.There are problems associated with each of these approaches. People whodiet are initially successful in losing weight. However, those whocomplete weight-loss programs lose approximately 10% of their bodyweight, only to regain two-thirds of it back within 1 year and almostall of it back within 5 years. Strict dieting alone results in loss oflean tissue as well as fat. When obese experimental animals are starvedto death, some preservation of fat, despite utilization of brain andheart for energy, is observed. Surgery involves many complications andis often only used in the morbidly obese as a last resort.

Drugs that are being developed can be broken down into three categories:drugs that target the brain, drugs that affect fat absorption by theintestines, and drugs that affect fat cells directly. Drugs that targetthe brain rarely affect only one CNS pathway. There are many potentialCNS side-effects, e.g., on memory, behavior, and sexual function inaddition to systemic effects (e.g., cardiac and pulmonary dysfunction).CNS appetite suppressant drugs share some of the drawbacks of dietaryapproaches including loss of lean as well as fat mass and lack of effecton specific fat depots. Drugs that affect fat absorption also tend toaffect absorption of other critical nutrients and often cause diarrheaor other gastrointestinal side-effects. These drugs also share some ofthe drawbacks of dietary approaches. Drugs that affect fat cellsdirectly (e.g., β3 agonists) have sometimes been developed initiallyusing animal models and later have proven to be less effective in humanswithout substantial expenditures for further development.

The group of drugs most likely to be effective in treating all forms ofobesity and with the best side effect profiles are likely to be drugsthat act directly on fat tissue to modify fat cell size or number.Currently, strategies to discover drugs acting at the level of the fatcell usually involve screening assays in animal models, animal aneuploidcell lines, or freshly-derived tissue from human subjects. Severalproblems are associated with these approaches including: (1) Fat cellsfrom humans are very different from rodent fat cells orpreadipocyte-like aneuploid cell lines. Consequently, drugs that appearpromising in such models have proven ineffective in human fat tissue.(2) Preadipocyte-like aneuploid cell lines exhibit a number ofdifferences from euploid cells. For example, 3T3-L1 cells containanywhere from a few to over 200 chromosomes, are immortal in culture,differentiate under conditions in which rat or human primary culturepreadipocytes do not, and exhibit differences from primary culturedpreadipocytes in the appearance of catecholamine responsiveness,capacity for de novo triglyceride synthesis, and hormone-sensitivelipase activity during differentiation. For these reasons, it has beenstated that findings in cell lines need to be verified in euploidpreadipocytes (MacDougald, O. A. et al. (1995) Annu. Rev. Biochem64:345-73; Smas, C. M. et al. (1995) Biochem J. 309, 697-710; Cornelius,P. et al. (1994) Annu. Rev. Nutr. 14:99-129) (3) Use of human fat cellsfor developing new drugs presents problems: sufficient human fat tissuefor assays is difficult to obtain, particularly in quantities sufficientfor high throughput assays, and fat tissue deteriorates rapidly.

Human preadipocytes are extremely valuable as a drug development systemfor the reasons given above, however, human preadipocytes have not beenused extensively to date due to a host of problems including (1)inability to obtain adequate amounts of fat tissue from which to isolatepreadipocytes (2) difficulty in inducing adipocyte differentiation (evenunder optimized differentiation culture conditions (Hauner, H., et al.(1989) J. Clin. Invest. 84:1663-70; Van de Venter, M., et al. (1 994) J.Cell. Biochem. 54:1-10), (3) inability to subculture human preadipocytesthat retain the capacity to differentiate (e.g., as compared to inprimary cultures). Thus there exists an art-recognized need for improvedmethods of obtaining human preadipocyte cells, in particular, forobtaining high yields of substantially pure preadipocyte cultures thatmaintain a high differentiative capacity.

SUMMARY OF THE INVENTION

The present invention features improved processes for isolating andculturing human preadipocytes. In particular, the invention featuresisolation processes for obtaining high yield, substantially pure humanpreadipocyte cultures and/or subcultures. Also featured are optimizedtechniques for differentiating human adipocytes. Human preadipocytes canthus be prepared in an inexpensive, consistent, and effective processyielding differentiated human preadipocytes in quantity. Humanpreadipocytes prepared according to the methodology of the instantinvention are particularly amenable for use in high-throughput drugscreening efforts.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention features high-yield, essentially pure, humanpreadipocyte and adipocyte cell populations and methods for obtainingsaid cell populations. In particular, the invention features methods forobtaining high yields of preadipocytes from a relatively limited sourceof human tissue, methods of culturing said preadipocytes such thatessentially pure cultures are obtained, and optimized methods ofdifferentiating said pure populations such that highly differentiatedadipocyte cultures are obtained. The preadipocyte and adipocyte culturesof the invention are particularly suited for use in high throughputscreening assays for identifying modulators of fat cell replication,differentiation and function. Preferred methods of the instant inventionfeature the clonal expansion and subsequent differentiation of humanpreadipocytes (or high yield preadipocyte populations) such thatreproducible high throughput screening assays can be carried out.

In one aspect, the invention features high-yield, essentially pure,human, preadipocyte populations. In another embodiment, the inventionfeatures highly-differentiated, human, adipocyte populations. In yetanother embodiment, the invention features methods of identifyingmodulators (e.g., inhibitors) of human preadipocyte/adipocytedifferentiation using the above-mentioned cell populations. Preferredmethods of identifying modulators (e.g., inhibitors) of humanpreadipocyte/adipocyte differentiation include assaying cell populationsfor a detectable fluctuation in fatty acid uptake (e.g., assaying cellpopulations for a detectable inhibition of fatty acid uptake).Modulators identified according to -the methodology of the instantinvention are also featured. Preferred modulators of the invention arethose that act directly on fat cells and/or fat tissue. Additionalpreferred modulators are those that act in a depot-specific manner.

Prior to describing the invention, it may be helpful to an understandingthereof to set forth definitions of certain terms to be usedhereinafter.

The term “preadipocyte” refers to a cell existing in or isolated fromfat tissue which is capable of replicating yet is committed to theadipogenic phenotype (i.e., is committed to differentiate into anadipocyte or fat cell). In their undifferentiated state, culturedpreadipocytes resemble fibroblasts (i.e., have a fibroblast-likemorphology). In particular, they exhibit a flattened, adherentmorphology and contain very little microscopically-detectable lipid. Theterm “human preadipocyte” refers to a preadipocyte existing in orisolated from human fat tissue.

The term “adipocyte” refers to a cell existing in or derived from fattissue which is terminally differentiated. In their differentiatedstate, adipocytes assume a rounded morphology associated withcytoskeletal changes and loss of mobility. They further accumulate lipidas multiple small vesicles that later coalesce into a single, largelipid droplet displacing the nucleus. The term “human adipocyte” refersto an adipocyte existing in or isolated from human fat tissue.

The term “partially-differentiated preadipocyte” refers to apreadipocyte which exhibits one or more markers of differentiation, forexample, accumulation of cytoplasmic lipid, but is still capable ofreplication.

The phrase “essentially pure” refers to a cell population, e.g., a humanpreadipocyte population, that has been isolated from its natural source(e.g., has been isolated or purified from fat tissue, for example, fromhuman fat tissue) and, through a purification step or series ofpurification steps, has been separated from other cells (e.g.,non-preadipocyte cells) and cellular debris. An essentially pure cellpopulation, as defined according to the instant invention, is at least90% pure, i.e., at least 90% of the cells are of the desired cell type(e.g., human preadipocytes) and less then 10% are contaminating (e.g.non-preadipocyte) cells. In a preferred embodiment, an essentially purecell population (e.g., an essentially pure preadipocyte population) isat least 95% pure. In a more preferred embodiment, an essentially purecell population (e.g., an essentially pure preadipocyte population) isat least 96%, 97%, 98%, 99% or 100% pure). Purity of a preadipocyteculture is most easily determined after the culture has beendifferentiated into adipocytes (i.e., by culturing for appropriate timesin the presence of differentiation-inducing and differentiationpromoting agents, as defined herein). Any marker of differentiation, asdefined herein, can be used to determine the percentage of cells thathave differentiated. For example, where 100% of the cells in a cultureof the invention have accumulated lipid droplets to an appreciable size(i.e. 100% of the cells are adipocytes), it can be concluded that 100%of the cells were preadipocytes of the original culture werepreadipocytes, i.e., the starting culture was 100% preadipocytes or 100%pure.

The phrase “capable of replication” is used to refer to cells (e.g.,preadipocytes or partially-differentiated preadipocytes) capable ofundergoing cell division. Cell division can be determined or assessedhistologically, by flow cytometry, indirectly (e.g., as an increase inDNA synthesis, for example, an increase in radiolabeled thymidine into acell or cell population).

The term “passage” refers to the transfer or transplantation of cell,with or without dilution, from one culture vessel to another. It isunderstood that any time cells are transferred from one vessel toanother, a certain portion of the cells may be lost and, therefore,dilution of cells, whether deliberate or not, may occur. The termpassage is synonymous with the term subculture. The number of passages apopulation can proceed through is characteristic of the quality of thepreparation.

The phrase “high-yield” means a yield of 10⁶ cells from 25 g or less ofisolated human fat, preferably a yield of 10⁶ cells from 10 g or less ofisolated human fat, more preferably a yield of 10⁶ cells from 5 g orless of isolated human fat, and most preferably a yield of 10⁶ cellsfrom 1 g or less of isolated human fat. The phrase “high yield” refersto the result of processes of the instant invention as compared topublished “low yield” processes, for example, processes yielding 10⁶cells from approximately 70 g of isolated human fat.

The phrase “enhanced differentiative capacity” means at least a 2-fold,preferably a 3, 4 or 5-fold, more preferably a 6-, 7-, 8-, 9-, 10-foldor greater improvement in extent of human preadipocyte differentiationbeyond established, published methods.

The phrase “differentiation-inducing agent” refers to a compound oragent that initiates or stimulates the differentiation of preadipocytesinto adipocytes. Preferred “differentiation-inducing agents” include butare not limited to insulin, insulin-sensitizing agents, substrates forlipid synthesis, PPAR ligands (e.g., natural ligands, for example,prostaglandin J₂, and synthetic ligands, for example,thiazolidinediones, and the like). The phrase “differentiation-promotingagent” refers to a compound or agent that enhances or accelerate thedifferentiation of preadipocytes into adipocytes. Preferred“differentiation-inducing agents” include but are not limited toinsulin, insulin-sensitizing agents, substrates for lipid synthesis,PPAR ligands, and the like.

“Differentiation-inducing agents” or “differentiation-promoting agents”vary considerably in effectiveness but share common effects on severalcellular signaling pathways including, but not limited to: (1) tyrosinekinase pathways (e.g., IGF-1-mediated tyrosine kinase pathway); (2)adenylyl cyclase/phosphodiesterase signaling pathways; (3)steroid/thyroid/peroxisome proliferator activated (PPAR)/retinoidnuclear receptors signaling pathways; and (4) protein kinase signalingpathways (MacDougald, O. A. et al. (1995) Annu. Rev. Biochem 64:345-73;Smas, C. M. et al. (1995) Biochem J. 309, 697-710; Cornelius, P. et al.(1994) Annu. Rev. Nutr. 14:99-129).

Following induction of differentiation through these signal transductionpathways, coordinated changes in the expression of over 600 genes occursleading to the acquisition and maintenance of the fat cell phenotype(MacDougald, O. A. et al. (1995) Annu. Rev. Biochem 64:345-73; Smas, C.M. et al. (1995) Biochem J. 309, 697-710; Cornelius, P. et al. (1994)Annu. Rev. Nutr. 14:99-129). These changes in differentiation-dependentgene expression are orchestrated by several transcription factorsincluding CCAAT enhancer binding proteins (C/EBPα, β, and γ), PPARγ, andothers (reviewed in MacDougald, O. A. et al. (1995) Annu. Rev. Biochem64:345-73; Smas, C. M. et al. (1995) Biochem J. 309, 697-710; Kirkland,J. L., et al.(1997) J. Amer. Geriatr. Soc. 45:959-67). Overexpression ofsome of these transcription factors, including C/EBPα and PPARγ, issufficient to induce the differentiation of preadipocytes (Lin, F. T.,et al (1994) Proc. Natl. Acad: Sci. USA 91:8757-8761; Hu, E. et al.(1995) Proc. Natl. Acad. Sci. USA 92:8956-60; Wu, Z., et al. (1995)Genes Defer 9:2350-63; Yeh, W. C., et al. (1995) Genes Devel. 9:168-81).

Markers of differentiation include (presented in order of detectablechanges in expression): (1) cytoskeletal genes; (2) lipoprotein lipase(LPL) and collagen isoforms; (3) adipocyte fatty acyl binding protein(aP2) and glycerol-3-phosphate dehydrogenase (G3PD); and/or (4) theinsulin sensitive glucose transporter (GLUT4), angiotensinogen (ang),apolipoprotein E (apoE), leptin, adipsin (complement factor D), proteinC3, factor B, and other genes occur that contribute to theendocrine/paracrine function of adipose tissue. Increased fat cell size,which is dependent on the balance between rates of lipogenesis (anddifferentiation) and lipolysis, is also a detectable marker of adipocyteor fat cell differentiation.

The term “fat depot” refers to a deposit of fat cells existing withinanimal, preferably human, tissue comprising essentially adipocytesand/or preadipocytes. Fat depots can be peripheral or visceral.Preferred fat depots include, but are not limited to subcutaneous fatdepots, omental fat depots and mesenteric fat depots. The phrase“depot-specific effect” or “fat depot-specific effect” refers to aneffect, for example a biological or physiological effect, that isparticular to one or more fat depots (or cells obtained from said depotor depots) but not common to fat cells or fat depots of all origins.

The term “mesenteric” means of or having to do with the mesentery. Themesentery is any membranous fold attaching various organs to the bodywall. Specifically, mesentery refers to the peritoneal fold attachingthe small intestine to the dorsal abdominal wall. For the purposes ofthe instant invention the term mesenteric is used to describe fat thatdevelops around the mesentery.

The term “omental” pertains to an area around the omentum. The omentumis a fold of peritoneum extending from the stomach to adjacent organs ofthe abdominal cavity. For the purposes of the instant invention the termomental is used to describe omental fat, or omental fat depots, whichdescribes fat that develops around the omentum.

The term “subcutaneous” is an art recognized term that refers to thelocation below the skin. In terms of this invention, the term refers tosubcutaneous fat, i.e., fat located in depots below the skin.

As used herein, a fat cell, preadipocyte or adipocyte “modulator” or“modulatory” compound or agent is a compound or agent that modulates atleast one biological marker or biological activity characteristic of fatcells and/or fat tissue. In preferred embodiments, compounds or agentsof the invention modulate at least one of (1) differentiation-specificgene expression, (2) lipid metabolism (e.g., lipogenesis and/orlipolysis), (3) fatty acid uptake, and/or (4) accumulation ofcytoplasmic lipid.

The term “serum-free” refers to a medium free of animal serum, free ofpartially-purified or characterized animal serums, serum substitutes andthe like. Preferably, a serum-free medium is free of bovine serum, fetalbovine serum, etc.

Where ranges are recited herein, all intermediate values within theranges are expressly within the scope of the invention.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Isolating and Subculturing of Human Preadipocytes:

The instant invention features methods of isolating and culturing orsubculturing human preadipocytes that are significantly improved overmethods previously published. In particular, the methods of the instantinvention feature isolation of essentially pure, high-yield, humanpreadipocyte cell populations that are capable of undergoing multiplerounds of division while maintaining differentiative capacity.Preadipocyte cultures of the instant invention maintain differentiativecapacity for at least 4 passages and preadipocytes capable ofdifferentiating after as many as 25 passages have now been obtained.This is quite significant when compared to published reports of humanpreadipocytes losing differentiative capacity after 3-4 passages. Thesignificant increase in replicative capacity ofdifferentiation-competent cells is due to a combination of the purity ofpreadipocyte cultures at outset (i.e., no “overgrowth” of contaminatingcell types) and the improved differentiation culture methodology of theinstant invention.

This ability to amplify preadipocytes that maintain differentiativecapacity results is key to being able to perform screening assays formodulators of adipocyte function. For example, even a 10-foldamplification (i.e., 10 passages) of human preadipocytes allowsexpansion of a single flask of cells into 1024 flasks. Adipocytesisolated and subcultured as described herein can further becryopreserved (“banked”) and passaged again after thawing. A summary ofthe preferred isolation methodology of the instant invention is asfollows:

-   -   A. Cells are isolated from tissue biopsies obtained during        surgery (e.g., abdominal surgery). Alternatively tissue, such as        for example omental fat tissue, may be obtained by laparoscopy.        Preferably, the time from tissue harvest to isolation is kept to        a minimum. In one embodiment, the time from tissue harvest to        isolation is no greater than 24 hours. In another embodiment,        the time from tissue harvest to isolation is no greater than 20        hours. In another embodiment, the time from tissue harvest to        isolation is no greater than 16 hours. In another embodiment,        the time from tissue harvest to isolation is no greater than 12,        8, 4 or 2 hours.    -   B. The tissue is transported from the operating room in a        buffered transport medium (e.g., a PBS buffered medium or        HBSS/sodium bicarbonate buffered medium) containing antibiotics        (e.g., gentamicin, amphotericin, penicillin, and/or        streptomycin) and L-glutamine.    -   C. The tissue is then sectioned into pieces between 5-10 mg and        placed in centrifuge tubes (e.g., 50 ml centrifuge tubes)        containing digestion medium. A preferred digestion medium is a        buffered medium (e.g., PBS or HBSS buffered medium) including        collagenase, BSA (e.g., fatty-acid free BSA), and, optionally,        L-glutamine. Preferably 3 mg of collagenase is used per gram of        tissue.    -   D. Omental tissue is processed first, since digestion of omental        tissue takes longer than mesenteric or subcutaneous tissue.        Subcutaneous tissue is processed next, followed by mesenteric.        The tissue is finely minced (pieces approximately 1-3 mm in        size) using sharp sterile scissors, then vortexed thoroughly and        placed in a shaking water bath at 37° C. (water level high        enough to submerge all of tissue in tubes).    -   E. The tubes are thoroughly vortexed every 10 minutes until the        tissue is fully digested.    -   F. The solution is then filtered through a gauze filter        (Steri-Pad 4×4 inch, Johnson and Johnson, NJ) and spun at 1000×g        for 10 minutes.    -   G. The resulting pellet is resuspended in an erythrocyte-lysing        buffer containing potassium bicarbonate, EDTA, and ammonium        chloride.    -   H. The tubes are placed in a shaking water bath at 37° C. for        five minutes and then spun at 1000×g for 10 minutes.    -   I. The pellet is then plated overnight in plating medium, which        consists of DMEM/F12, HEPES, sodium bicarbonate, penicillin,        streptomycin, L-glutamine, and NuSerum (a semi-artificial serum        supplement available from BD BioSystems, Inc., Bedford, Mass.).        (Alternatively, cells (e.g., cells obtained from laparoscopy)        can be plated in α-MEM-based plating medium as follows. Cells        are plated in a 100 mm cell culture dish for a minimum of 16        hours, and not more than 24 hours, at 37° C., 5% CO₂ in α-MEM 10        medium (alpha MEM sodium bicarbonate buffered medium        supplemented with 10% fetal bovine serum and antibiotics,        streptomycin and penicillin, see Table 7) prior to subsequent        processing in artificial, substantially animal product free        medium (such as for example, NuSerum available from BD        BioSciences, cat. # 35-5504) in order to obtain optimum yield.)    -   J. After 6-24 hours of adherence, the plates are then washed and        trypsinized. Preferably the cells are allowed to adhere for        16-24 hours prior to trypsinization, more preferably for about        18 hours prior to trypsinization.    -   K. The trypsinized cells are centrifuged at 1000×g for 10        minutes, and the resulting pellet is resuspended in DMEM/F12        plating medium (as described in Table 10). The cell density is        determined using trypan blue and a hemocytometer, counting all        four quadrants. # of cells =(cell count/4)×2×(1×10⁴)×(# ml cells        are suspended in).    -   L. The cells are plated at a density of5.0×10⁴cells/cm², e g.,        in T-25 flasks. The cells are grown to confluence and either        passaged 1:2 for further cell replication, frozen in liquid        nitrogen storage, or further cultured in differentiation medium.    -   M. When passaging cells, preadipocytes will have a        fibroblast-like appearance. During the isolation procedure, it        is possible to have contaminating cells present, especially in        omental preadipocyte preparations. These contaminating cells are        flat and have a rounded morphology, and in many cases have a        raised circular center, giving these cells a “fried egg”        appearance. The percentage of contaminating cells can range from        zero to as much as 50%, in rare cases. These cell types are        removed during the passaging process when the preadipocyte        population reaches greater than 75% confluency. The        contaminating cells are removed by differential trypsin        treatment. The dish or flask of adhered cells is washed once        with PBS/EDTA (Table 8), then trypsin in PBS/EDTA (Table 9) is        added for approximately one minute, with brief swirling every 20        seconds. The trypsin solution is then removed, and remaining        attached cells are washed again in PBS/EDTA. The cells are then        detached by addition of trypsin in PBS/EDTA, and are replated        into a new dish or flask of similar size and allowed to grow. If        contaminating cells are still present after a few days of cell        growth, the procedure is repeated when the preadipocyte        population is greater than 75% confluency.

II. Differentiation of Human Preadipocytes into Adipocytes:

The differentiation methods of the instant invention result indramatically improved human adipocyte cultures. In particular, humanadipocytes cultured according to the methods of the instant inventionare uniform and highly differentiated. Moreover, the isolation anddifferentiation methods of the invention are suitable to isolation offat from each of visceral, omental fat and mesenteric fat depots.Compared to published methods, cultures of omental adipocytes isolatedand cultured according to the methods of the instant invention exhibitat least 4-fold enhanced differentiation (as determined according to thefatty acid uptake assay described herein) and cultures of visceraladipocytes exhibit at least 100-fold enhanced differentiation. A summaryof the preferred differentiation methodology of the instant invention isas follows:

-   -   A. Preadipocytes are seeded in PM at 3×10⁴ cells/cm2 to insure        100% confluency upon adherence to the cell culture dish or        plate.    -   B. 48 to 120 hours after seeding (optionally with a changing of        PM every 2-4 days), plating medium is removed and cells are        exposed to a serum-free differentiation medium (DM) consisting        of the following basic components:

DMEM/F12, HEPES, sodium bicarbonate, penicillin, streptomycin,L-glutamine, biotin, human insulin, panthothenic acid, dexamethasone,and triiodo-L-thyronine. Exemplary concentrations are as follows:

-   -   -   DMEM:F12 (1:1),        -   15 mM HEPES,        -   15 mM NaHCO3,

    -   2 mM glutamine,        -   33 μM biotin,        -   0.5 μM insulin,        -   17 μM pantothenate,        -   0.1 μM dexamethasone,        -   2 nM triiodothyronine, and        -   antibiotics.        -   In other embodiments, cells are exposed to differentiation            medium for two to ten days or two to fourteen days.

    -   C. In addition, a number of other factors are preferably        included in the DM at various concentrations, for example        transferrin, fetuin, rosiglitazone, and isobutylmethylxanthine        (IBMX). Exemplary concentrations are as follows:        -   the thiazolidinedione rosiglitazone (up to 1 μM),        -   fetuin (up to 1 g/L),        -   transferin (up to 10 mg/L), and        -   isobutylmethylxanthine (up to 600 μM).

    -   D. The medium is changed every two to three days as        preadipocytes differentiate into adipocytes. After cells have        attained a rounded morphology, DM can be changed to DM without        IBMX. After cells have become more rounded and/or triglyceride        deposits become visible (i.e., microscopically visible), DM        without IBMX can be changed to DM without IBMX, rosiglitazone,        insulin or dexamethasone.

The isolation/differentiation method described in Examples 1 and 2 wascompared to a generally-used, published method (Hauner, H., et al (1989)J. Clin. Invest. 84:1663-70; Hauner, H., et al. (1995) Eur. J. Clin.Invest. 25:90-96)). The published method (as most recently modified)involves collagenase digestion (with FBS), filtration, treatment with anerythrocyte lysis buffer, and plating in a medium that contains FBS.Confluent cells are then exposed to a differentiation medium thatcontains: DMEM:F12 (1:1), 15 mM HEPES, 15 mM NaHCO₃, 33 μM biotin, 0.5μM insulin, 17 μM pantothenate, 0.1 μM dexamethasone, 0.2 nMtriiodothyronine, and antibiotics. The methodology of the presentinvention differs in many respects from the published method. Forexample, different mincing procedures are used for different fat depots.BSA is used in the collagenase solution both to increase yield andbecause exposure to FBS inhibits subsequent differentiation of humanpreadipocytes. Different filtering material is used. As in the publishedmethod, digests are treated with an erythrocyte lysis buffer, sinceprolonged exposure to erythrocytes inhibits preadipocytedifferentiation. Cells are plated in a medium containing asemi-artificial serum supplement instead of FBS to prevent effects ofexposure to FBS on subsequent differentiation. Plating density isdifferent. Cultureware was selected that enhances yield anddifferentiation. Unlike the published method, human preadipocytes aretrypsinized and are replated after 18 hours. It has been determined thatwithin about 18 hours after the initial plating, contaminating cells(e.g., macrophages mesothelial cells) strongly adhere but adipocytesonly loosely adhere. Subsequent mild trypsination results in detachmentof preadipocytes but not contaminating cells, increasing culture purity.The trypsination/replating step also facilitates accurate cell counting(i.e., non-cell contaminants, tissue debris, cellular debris (e.g., cellmembranes) and the like are removed).

Cells are plated to insure confluence. Confluent cells are exposed to aserum-free differentiation medium that contains the same basiccomponents as the medium used in published methods (although atdifferent concentrations) as well as a number of other factors,including thiazolidinediones, fetuin, transferin, andisobutylmethyl-xanthine pretreatment, among others. Serum free-medium isused as it has been determined that the published serum-containing mediainhibit the differentiation of human adipocytes.

Although some of these approaches have been used in rodent preadipocytecell lines and, in some cases, human preadipocytes, it is thecombination of protocol modifications that results in the instanteffective and rapid method for differentiating human preadipocytes.

Unlike the published method, 3^(rd), 4^(th), or later passage cells areroutinely used, thus greatly increasing yield. Sufficientdifferentiation of 1 0th passage cells is obtained that signal-to-noiseratio is satisfactory even in these cells. The published method is quiteineffective for cells that have been subcultured (Entenmann, G., et al.(1996) Am J. Physiol. 270:C1011-6)). Typically, primary culture cellsneed to be exposed to the published differentiation medium for over 18days for cells to accumulate lipid droplets. In fourth subculture humanpreadipocytes, cells differentiated under the conditions describedherein accumulate extensive lipid within 10 days. Parallel treatment ofsimilarly subcultured cells with the published method resulted in verylittle adipogenesis.

Advantageously, isolated depot specific preadipocytes may becryopreserved until thawed for use. For example, after isolation andgrowth as described above, preadipocytes are detached from the flask ordish using trypsin in PBS/EDTA. Cells are spun down and resuspended inice-cold freezing medium (DMEM/F12, HEPES and bicarbonate bufferedmedium plus 10% glycerol), placed at −70° C. overnight and thentransferred to liquid nitrogen for storage. For subsequent use, cellsmay be thawed in a water bath at 37° C., then slowly diluted in platingmedium to lessen/prevent osmotic shock. Cells may be spun down to removethe glycerol and then resuspended in plating medium for culture, oralternatively allowed to adhere for 4 to 6 hours at 5% carbondioxide/37° C., then changed with fresh plating medium for culture.

III. Screening Assays:

The human preadipocyte and/or adipocyte populations of the instantinvention are particularly amenable to use in high-throughput drugscreening efforts to identify compounds that act directly on fat cells(and/or their targets). In particular, the human preadipocyte and/oradipocyte populations of the instant invention are useful in assayingfor agents that modulate the lipid content of adipocytes or fat cells(i.e., agents that modulate fatty acid uptake and/or lipid accumulation)and in assaying for agents that modulate adipocyte or fat celldifferentiation. Preferably the human preadipocyte and/or adipocytepopulations of the instant invention are used in assays to identifyagents that inhibit the lipid content of adipocytes or fat cells (i.e.,agents that inhibit fatty acid uptake and/or lipid accumulation) and/orin assays to identify agents that inhibit differentiation of adipocytesor fat cells. Compounds so identified are particularly useful for thedevelopment of drugs which directly act on fat tissue in vivo.

Accordingly, the present invention features rapid high-throughputscreening assays or methods (“HTS assays”) for identifying compounds oragents that effect or modulate human preadipocyte and/or adipocyte fattyacid incorporation and/or differentiation. Sensitive and reproducibleHTS assays featuring human preadipocyte and/or adipocytes are possibleas a result of (1) the improved methods for isolating and purifyinghuman preadipocytes; (2) inexpensive, consistent, and effective methodsyielding differentiated human preadipocytes in quantity; and (3) methodsdeveloped for sensitive measurement of changes in fatty acid content.The improved isolation, culture and differentiation methodology of theinstant inventions also make it possible to established a “bank” ofstored (i.e., cryopreserved) human preadipocytes such that a steady andreproducible supply is available.

Hits (e.g., lead compounds) identified as described herein can furtherbe subjected to a variety of secondary screening assays. In certainembodiments, secondary screening assays are utilized to validate a hitor lead as a fat modulatory compound (e.g. an effector of fataccumulation). In other embodiments, secondary screening assays areutilized to determine pathways affected by a hit (e.g., lead compound)and/or to identify a target (e.g., cellular or molecular target) of thehit or lead.

Targets in the pathway(s) resulting in adipogenesis, fatty acid uptakeand/or triglyceride synthesis/storage (lipogenesis), or triglyceridebreakdown (lipolysis) and/or fatty acid oxidation may be also beidentified using the methods provided herein. Target identificationallows rational design of agents and drugs useful for preventing orreversing obesity. Identification of an agent and its target allowidentification of effector sites, facilitating design of more effectivedrugs.

Preferred features of the screening assays of the instant invention areconsistency and sensitivity.

A. Test Compounds

Test compounds include small molecules, peptides, polypeptides andnucleic acid molecules or libraries of such molecules. The testcompounds of the present invention can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; and synthetic library methods using affinitychromatography selection.

Libraries of compounds may be presented in solution, or on beads orchips or the like. Exemplary libraries include, but are not limited tosynthetic chemical product libraries and natural product libraries(e.g., partially deconvoluted natural product libraries).

B. High Throughput Screening Assay Formats

B 1. Identification of Lead Compounds

The high-throughput screening assays (“HTS assays”) for theidentification of compounds or agents that modulate fat cell activity,function or phenotype have been developed featuring either humanpreadipocytes, partially-differentiated adipocytes or adipocytes,isolated and cultured as described above. The HTS assays preferablyfeature identifying compounds that exhibit an effect on fatty aciduptake/accumulation according to the fatty acid-based celluomic assaydescribed herein (see Example 4). Although cell-based screens are moretechnically demanding than target-based screens, this approach offersseveral advantages as a primary screen. Use of specific targets (i.e.,molecular targets or pathways) obviously narrows the range of effectivetherapeutic classes likely to be discovered, while potentially novelcompounds acting on unknown targets can be discovered using a cell-basedapproach. Moreover, no information is yet available about moleculartargets likely to result in discovery of fat depot-specific anti-obesityagents in the human, and the time, effort, and risk involved inidentifying cellular targets is considerable. Indeed, the cell-basedapproach may lead to identification of novel targets for use in laterscreening efforts. By allowing the human adipocyte to “select” the geneand protein target, the drug discovery process will be accelerated.

In certain assay formats, preadipocytes are contacted with a testcompound in the presence of differentiation medium, cultured for apredetermined amount of time in the presence of the test compound andthe differentiation medium (e.g., for an amount of time sufficient toallow differentiation into adipocytes) and assayed at the end of thepredetermined time for fatty acid uptake/accumulation. The skilledartisan will appreciate, however, that differentiation of adipocytes isnot essential to the methodologies of the instant invention. In certainembodiments, compounds that result in a lessening or reduction of fattyacid uptake/accumulation are selected as potential lead compounds or“hits”. Such compounds are useful, for example, as therapeutic agents(or in the development of therapeutic agents) for effecting weight loss,treating obesity, etc. In other embodiments, compounds that result in anincrease of fatty acid uptake/accumulation are selected as potentiallead compounds or “hits”. Such compounds are useful, for example, astherapeutic agents (or in the development of therapeutic agents) for thetreatment of cachexia and/or anorexia. Additionally, hits or leadsidentified according to the methodologies of the present invention maybe useful, for example, as therapeutic agents (or in the development oftherapeutic agents) for the treatment of reduced insulin sensitivity,insulin resistance, diabetes (e.g., Type II diabetes), and the like.

In other assay formats, human adipocytes (in differentiation medium) arecontacted with a test compound, cultured for a predetermined amount oftime in the presence of the test compound (e.g., for an amount of timesufficient to allow loss of lipid from lipid droplets orde-differentiation of adipocytes) and assayed at the end of thepredetermined time for fatty acid uptake/accumulation. Compounds thatresult in a lessening or reduction of fatty acid uptake/accumulation areselected as potential lead compounds or “hits”.

HTS assays are preferably carried out using 96-well or 384-well assayplates, although other configurations (e.g., other multi-well formats orcoverslip formats) are within the scope of the instant invention.

In preferred embodiments, compounds or agents are screened for bothtoxicity (e.g., in vitro toxicity) and in vitro biological activity.Non-toxic compounds (e.g., those that are non-toxic at the at the lowmicromolar level) (potency (ED₅₀)) exhibiting in vitro biologicalactivity (e.g., efficacy (maximum response)) are preferred. Toxicityscreens can be performed utilizing preadipocytes or adipocytes isolatedaccording to the present invention. Alternatively, compound toxicity canbe screened using non-adipocyte cells, for example, using fibroblasts orhepatocytes. Toxicity screens using preadipocytes or adipocytes can beperformed prior to biological activity assays, subsequent to biologicalactivity assays, or in parallel with biological activity assays (i.e.,in parallel cultures). In a preferred embodiment of the instantinvention, toxicity screens using preadipocytes or adipocytes areperformed in the same cultures as biological activity screens (see e.g.,Example 6 and 7). Acceptable toxicity of the agent or modulator is lessthan 20% in the cell assay, more preferably the toxicity is less than15%, most preferably the toxicity is less than 10%.

The two formats are designed to identify hits that potentially targetdifferent stages of adipogenesis. Hits detected after early addition mayaffect differentiation and/or fat accumulation, while those detectedfrom late addition may affect lipogenesis or lipolysis. Therefore,different classes of drugs are likely to result from this screeningstrategy. Preferably, “hits” (or positive scoring library components)from this assay will have a response of ≦50% of the control value at 3μMsynthetic compound or 0.05× dilution of natural product with minimaltoxicity in the cell-based toxicity assay.

Preferably, hits are automatically flagged (plate number, row, andcolumn) with the instrument control and data analysis software. Librarycomponents are preferably screened in triplicate, and are checked forfluorescent properties that could interfere with interpretation of assayresults. Potential false positives from this screen could arise througha variety of mechanisms. For example, compounds that interact with thefluorescent fatty acid itself and block its uptake could give a positiveresponse in this assay. Also, compounds that are sequestered in fatdroplets with the fluorescent fatty acids and that quench theirfluorescence could result in false positive outcomes. In addition tofalse positives, hits arise by targeting undesired mechanisms. Compoundsaffect mechanisms other than differentiation, lipogenesis, or lipolysis,or can be toxic to preadipocytes, but not to fibroblasts.

A preferred primary screening protocol is as follows.

-   -   A. A high-throughput screen (HTS) technique for discovering        compounds that modulate the differentiated preadipocyte or        adipocyte's ability to take up and/or accumulate a        fluorescently-labeled fatty acid (FA*) is featured. The assay        features differentiated human preadipocytes, but the procedure        can be modified in a number of ways to discover novel compounds        affecting the adipocytes of human and other species (i.e.,        rodent). For example, the day of compound addition during        preadipocyte differentiation process can be changed as well as        the incubation period. Similarly, the day of FA* addition, its        type of fluorescent label and chain length, and its        concentration and incubation time can be modified.    -   B. Up to five cell strains of frozen primary human subcutaneous        preadipocytes (e.g., subcutaneous preadipocytes) are used, but        this number can be increased as needed. Omental and mesenteric        preadipocytes can be used in a similar manner. Cells are 10        thawed in PM and grown in tissue culture flasks under standard        incubation conditions (5% carbon dioxide, 37° C.). Cells are        passaged 1:2 upon 80-100% confluency. Cells are passaged up to        ten times to produce enough cells for primary screening.    -   C. Cells are seeded and differentiated, as described in the        previous section, in 384-well plates.    -   D. On day 6 of preadipocyte differentiation, the DM is first        changed as described in the previous section. Cells are then        incubated in a replicate manner (i.e., in triplicate) with        compounds. Compounds are typically dissolved 100% DMSO, but can        be dissolved in a different solvent or aqueous solution.        Compounds are further diluted in a PBS or similarly buffered        solution in the absence or presence of albumin to aid compound        solubility. After extensive mixing, the diluted compound is        added to cells; for compounds initially dissolved in 100% DMSO,        the final concentration of solvent in the cell solution is 0.1%.    -   E. Negative control cells are treated with the equivalent        compound solvent or solution diluted in the compound diluent        buffer (i e., for compounds dissolved in 100% DMSO and diluted        in a PBS/BSA buffer, the negative control cells are treated in a        similar manner without compound to give a final concentration of        0.1% DMSO). Positive control cells are treated with a compound        or reagent known to modulate their ability to take up and/or        accumulate fatty acids. For example, carbonyl cyanide        p-(trifluoromethoxy)-phenylhydrazone (FCCP) is a potent        uncoupler of oxidative phosphorylation in mitochondria, and        inhibits FA* uptake and/or accumulation in a dose-responsive        manner.    -   F. On day 9 of preadipocyte differentiation, FA* is diluted into        a PBS or similarly buffered solution in the absence or presence        of albumin to aid its solubility. See Table 11 for the current        fatty acid buffer (FAB) used. The FA* currently used is        4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic        acid (C₁-BODIPY® 500/510 C₁₂, catalog # D-3823, Molecular        Probes, Eugene, Oreg.). FA* stock concentration is 10 mM in 100%        DMSO, frozen at −20° C.    -   G. Cell plates are pre-washed with FAB, preferably three times,        to remove unincorporated compound, and then FA* is added in FAB        at 10 uM final concentration. Cells are incubated for four hours        at 5% carbon dioxide/37° C., then post-washed with FAB,        preferably three times, to remove unincorporated FA*. Cellular        fluorescence of triglyceride droplets that have incorporated FA*        is measured on a microplate reader. Efficacy of compounds on        inhibiting FA* accumulation is determined by:        % Efficacy=100−(sample fluorescence/negative control        fluorescence×100).    -   H. Subsequent determination of compound toxicity on cells is        measured by incubating cells (after the post-wash and FA*        reading) with an aliquot of Alamar Blue (catalog # DAL-1100,        BioSource International), a fluorescent dye that is reduced via        cellular metabolism. After incubation of cells with the dye for        up to four hours, fluorescence of the reduced compound is read.        Alternatively, compound toxicity on cells can be determined in        an experiment separate from the primary screen        uptake/accumulation assay. Toxicity of compounds is determined        by:        % Toxicity =100−(sample fluorescence/negative control        fluorescence×100).

B2. Pathway and/or Target Identification

Secondary assays may be run on identified “hits” to define thepathway(s)/mechanism of action on which such an agent/hit may act. Thesepathway(s) comprise four categories: Adipogenesis (key transcriptionfactors and enzymes that function in the differentiation ofpreadipocytes to adipocytes), lipogenesis (fatty acid uptake and/ortriglyceride synthesis/storage), lipolysis (triglyceride breakdown), andoxidation (fatty acid metabolism). Preferably, only hits exhibitingacceptable toxicity limits are pursued. Pathways affected by the agentsmay be identified using assays well known to those of skill in the art.

Pathways related to adipogenesis may be investigated by measuringexpression of related transcription factors such as those encoded by thePPAR gene family (e.g. PPARγ), and the C/EBP gene family (e.g. C/EBPα,C/EBPβ) and/or the transport protein GLUT4 gene and/or the aP2 gene.Such transcription factors and other proteins may be assayed usingWestern analysis using an appropriate antibody and extracts of cells asdescribed by Ausubel and Brent; Short Protocols in Molecular Biology,4^(th) Edition, 1999, John Wiley & Sons, Inc.). Preadipocytes ordifferentiated adipocytes may be exposed at various times to an agent invehicle, then extracts made and run in a western blot assay. Anti-GLUT4antibodies are available from Santa Cruz Biotech. Inc., (Santa Cruz,Calif.) and from Alpha Diagnostic International, (San Antonio, Tex.).PPAR antibodies are available from Research Diagnostics, Inc., Flanders,N.J.). C/EBP as well as PPAR antibodies are available from Active Motif,Carlsbad, Calif. Anti-aP2 antibodies may be obtained from Dr. D. A.Bernlohr at Univ. of MN. The reactive bands may be visualized using anenhanced chemoluminescence system (Amersham, Oakville, Ontario).Additionally, glycerol-3-phosphate dehydrogenase (G3PD) activity and/orPPAR gamma activation (e.g., Jeppesen et al, U.S. Pat. No. 6,468,996;Smith, U.S. Pat. No. 6,294,559) may be measured. G3PD activity may bemeasured as described by Sottile and Seuwen (2001) AnalyticalBiochemistry 293:124-128.

With respect to adipogenesis, adipocyte fat droplets are composedpredominantly of triglycerides, which in turn are made up of fatty acidchains bound to a glycerol backbone. Active lipogenesis may beinvestigated by monitoring the conversion of radiolabeled glucose intothe glycerol backbone, or of fatty acids such as palmitate or oleateinto tri-, di-, and mono-glycerides; quantitation may be done via thinlayer chromatography (TLC) to separate the lipid components, andsubsequent scintillation counting of the desired TLC spot. Totaltriglyceride levels may be performed using commercially available kits,such as Triglyceride E kit from Wako (Osaka, Japan) or Infinity glycerolmeasurement kit from Sigma (St. Louis, Mo.), or by quantitating theamount of Oil Red O (Sigma, St. Louis, Mo.) staining of fat droplets inadipocytes.

Lipolysis may be investigated by measuring the release of glycerol orfatty acids from the fat droplets of adipocytes into the mediumenvironment (eg. U.S. Pat. No. 6,096,338; U.S. Pat. No. 6,509,480).Glycerol may be measured using the techniques described above forLipogenesis. Released fatty acids may be measured by preloading cellulartriglycerides with a fluorescent fatty acid and then by monitoringdifferences in fluorescence under conditions favoring lipolysis. Inaddition, binding of fatty acids to ADIFAB (acrylodated intestinal fattyacid binding protein (ADIFAB); Molecular Probes, Eugene Oreg.) alter itsfluorescence spectrum, which can be quantified to measure fatty acidrelease (Richieri, et al, 1992, J Biol Chem 267(33):23495-501; Richieriet al, 1994 J Biol Chem 269(39):23918-30; Richieri et al, 1999, Mol CellBiochem 192(1-2):87-94).

Fatty acid oxidation in adipocytes may be investigated by monitoring theconversion of a tagged fatty acid (i.e., 14-C labeled fatty acid (i.e.,oleate or palmitate)) to carbon dioxide in a closed system environment.In addition, oxygen consumption, reflective of cellularmetabolism/oxidation, may be measured with an oxygen electrode such as aBiological Oxygen Monitor, MODEL 5300 (YSI Inc., Yellow Springs, Ohio,U.S.A.)). Alternatively, oxygen levels in the medium surrounding cellsmay be quantitated using an oxygen binding fluorescent probe (BDBioScience, Bedford, Mass.; Oxygen Biosensor System (OBS)).

Compounds having activity in one or more secondary screens (i.e.,compounds effecting or acting via various pathways, may have particularusefulness based on the nature of the pathway. For example, compoundseffecting adipogenesis (e.g., inhibiting differentiation) may haveparticular usefulness as therapeutics (or in the development oftherapeutics) for use in pre-diabetic and/or post-liposuction control ortreatment. Compounds effecting lipogenesis (e.g., inhibiting ordecreasing triglyceride synthesis) may have particular usefulness astherapeutics (or in the development of therapeutics) for use inmaintaining weight, for example, after weight loss. Compounds effectinglipolysis (e.g., increasing fat release) may have particular usefulnessas therapeutics (or in the development of therapeutics) for use inreducing central fat. Compounds effecting oxidation (e.g., increasingfat burning) may have particular usefulness as therapeutics (or in thedevelopment of therapeutics) for use as diet aids, for example, duringearly weight loss or pre-gastric bypass.

D. Fat Depot-Specific Screens

Drugs that target visceral (mesenteric and omental) fat are ofparticular interest, since the accumulation of visceral fat (e.g.,omental and/or mesenteric fat) carries a greater risk of morbidity andmortality than peripherally distributed fat (Rosenbaum, M., et al.(1997) New Engl. J. Med. 337:396-407). In particular, a high ratio ofvisceral to subcutaneous fat has been identified as a key risk factorfor cardiovascular disease. Moreover, hypertrophy of visceral fat hasbeen implicated in metabolic disorders, for example, the metabolicsyndrome, Syndrome X.

Considerable evidence, both published and in preliminary studies,indicates that there are sufficient differences between subcutaneous andvisceral preadipocytes to suggest that hits that primarily target onetype of fat depot will be found. Significant differences exist in geneexpression between the two cell types, glucocorticoid receptors andleptin, for example. Insulin action also appears to differ betweenomental and subcutaneous adipose tissue (Arner, P. (1997) Journal ofEndocrinology 155:191-2), and, in a possibly related finding,preadipocytes from the two depots (from the same individuals) show adifference in susceptibility to the differentiation-promoting effects ofPPARγ agonists.

C Lead Compound Characterization and/or Optimization

Lead compounds, also referred to herein as “hits”, exhibit biologicalactivity detectable as an effect of fat cell, preadipocyte or adipocytereplication, differentiation or function. Preferably, lead compounds or“hits” exhibit a measurable or appreciable effect on replication,differentiation or function while being non-toxic to fat cells,preadipocytes or adipocytes. Lead compounds or “hits” preferably exhibita potency of at least 500 nm, preferably at least 200 nM, morepreferably at least 100 nM, more preferable at least 75 nM, even morepreferably at least 50 nM, and even more preferably at least 10 nM. Fatdepot specific lead compounds or hits preferably exhibit of at least3-4-fold, more preferably at least 5-6-fold, more preferably at least7-, S-, 9- or 10-fold.

IV. Pharmaceutical Compositions

Compounds identified according to the methodology of the instantinvention can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the nucleicacid molecule, protein, antibody, or modulatory compound and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Pharmaceutical compositions comprising compounds identified according tothe methodology of the instant invention are particularly useful for thetreatment of diseases and/or disorders including, but not limited to,obesity, diabetes, insulin-resistance, hyperinsulinemia, hyperglycemia,hyperlipidemia, weight regulation disorders, eating disorders, cachexia,anorexia and the like.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference.

EXAMPLES Example 1 Isolation of High Yield, Essentially Pure HumanPreadipocytes

This example describes methods of isolating a high yield, essentiallypure population of human preadipocytes. The methods provided herein aresuitable for the isolation of preadipocytes from subcutaneous,mesenteric and omental fat depots. At least six parameters ofart-recognized adipocyte isolation procedures were varied and optimalconditions are described. Use of the optimized methods described hereinresults in reduced costs, time, and tissue requirements associated withhuman adipocyte screening assays. Currently, commercial isolationprocedures for human preadipocytes require up to 70 g subcutaneous fattissue (obtained by liposuction) to isolate 10⁶ cells. The preadipocytesource is further limited to only subcutaneous fat. Utilizing themethods disclosed herein, it is now possible to obtain 10⁶ cells from 1g or less of human abdominal subcutaneous fat and further, to obtainsuch yields from both mesenteric and omental fat.

In isolating preadipocytes from fat tissue, fat tissue is transported tothe laboratory, minced, digested in a collagenase solution, filtered,and plated. Each of these steps were varied to optimize the yield andquality of cultures. Yield was determined by calculating the number ofpreadipocytes recovered per gram of fat tissue, by counting cells at thedifferential replating step used to remove potentially contaminatingcell types. Examples of parameters to be varied to enhance yield arepresented in this example.

Decreasing Time Between Fat Tissue Harvest and Tissue DissociationEnhances Preadipocyte Yield

Because fat cells are very fragile and have limited viability ex vivo,the effect on yield of isolating preadipocytes from fat tissue samplesshortly after surgery, after 24 h, and after 7 d was determined. The fatsamples were maintained at room temperature in a transport medium thatcontains essential nutrients and antibiotics. Preadipocyte yielddecreased 22% within the first 24 hours following fat tissue harvest(53±4 vs. 42±4×10⁴ cells/g from fat tissue digested shortly aftersurgery and after 24 h, respectively; N=9; p=NS by Duncan's multiplerange test). Within a week following fat tissue harvest, yield decreased90% (53.1±4.2 vs. 5.3±0.6×10⁴ cells/g from fat tissue digested shortlyafter surgery and after 72 h, respectively; N=9; p<0.01 by Duncan'smultiple range test). Overall, increasing time between surgery anddigestion to isolate preadipocytes resulted in decreasing yield(p<0.005; ANOVA). Thus, while preadipocytes are more hardy than fatcells, presumably to permit fat tissue regeneration, yields are improvedby rapid processing. Preferably, tissue dissociation (e.g., mincingand/or digestion) is accomplished within the first 24 hours followingfat tissue harvest (e.g., surgery).

Modification of Mincing Technique Based on Fat Tissue Source EnhancesPreadipocyte Yield

In comparing coarsely minced fat tissue to finely minced tissue beforecollagenase digestion, preadipocyte yield was higher in coarsely thanfinely minced abdominal subcutaneous (yield 120% higher) and mesenteric(80% higher) tissue. Conversely, yield was 7% higher in omental tissuethat had been finely versus coarsely minced. Hence, modifications ofthis step in the preparation of preadipocytes have depot-specificeffects on yield. This finding has been incorporated into the proceduresused to isolate cells from different depots.

Modification of Media Components Enhances Preadipocyte Yield

Improved preadipocyte yield from human fat tissue has been found uponadding fetal bovine serum (FBS) to collagenase solution. Therefore,various types and concentrations of serum were used to optimize yield.Certain types of sera (e.g., Nuserum) improved yield, and this occurredin a depot-specific manner with the greatest improvements in yieldachieved in omental tissue. Several serum components, such as albumin oranti-protease activity, could have contributed to this effect. Albuminand anti-protease effects were independently tested.

Bovine serum albumin (BSA) had a substantial effect on omentalpreadipocyte yield in particular. Yield was improved 1.5±0.2 fold from91±16 to 123±16×10⁴ cells/g of omental fat tissue (p<0.02; paired Ttest; N=8). Interestingly, BSA had no 30 significant effect on abdominalsubcutaneous or mesenteric preadipocyte yield, so that under theseconditions, yield/g of tissue was higher from omental than subcutaneousor mesenteric fat (p<0.01; Duncan's multiple range test; N=8). Sincemore subcutaneous than omental fat is available from each patient, thisapproach for augmenting omental preadipocyte yield has enhanced thecapacity to screen for agents with depot-specific effects.

Collagenase preparations contain some trypsin activity. To test if yieldcould be enhanced by augmenting the trypsin activity of collagenase,various amounts of trypsin were added to the collagenase solution.Trypsin supplementation caused a decrease in yield. Because of this andbecause serum, which enhances yield when added to collagenase inhibitstrypsin activity, effects of adding trypsin inhibitors to thecollagenase solution were determined. These agents caused a reduction inyield. Hence, an optimized collagenase solution including BSA ratherthan serum was developed.

Donor Characteristics have No Significant Effect on Preadipocyte Yield

Effects of donor characteristics on preadipocyte yield and digestiontime were determined to assist in subject selection. In particular, bodymass index (BMI; weight/height²), age, gender, and ethnicity on fattissue digestion time and preadipocyte yield in mesenteric, omental, andabdominal subcutaneous fat specimens from 72 subjects was determined.Using applied regression analysis, it was determined that digestion timeincreased with donor age (p<0.05) and was longer for omental thansubcutaneous, and subcutaneous than mesenteric fat (p<0.0001). However,host characteristics had no significant effect on preadipocyte yield.

Choice of Filter Enhances Preadipocyte Yield

After collagenase digestion, filters are used to remove undigestedconnective tissue. The effects of several different types of filters onyield were determined and it was found that gauze filters gave the bestresults. For example, 100 mesh nylon filters resulted in a 2.7 foldlower yield than gauze filters (p<0.0005; unpaired T test with pooledestimate of variance ; N=65).

Choice of Cell Culture Surface Enhances Yield (and Extent ofDifferentiation)

The effects of a number of plate types and plate coatings on yield anddifferentiation was determined. For example, the yield and/or extent ofdifferentiation was reduced by the following products, compared touncoated plates: poly-D-lysine, collagen type IV, and laminin. Based onthese studies, optimal plates and coatings for yield anddifferentiation, were determined.

Optimization of parameters described in this example results in agreater than 3-fold improvement in yield as compared to previouslypublished methods. In particular, it is possible to obtain one millioncells from 1 g of fat tissue (or less). Moreover, these cells can bepassaged at least 34 times before replicative arrest (noting fat-depotdependent differences) and the cells are further able to be maintainedin the differentiated state for over 5 months (see below).

Example 2 Methods of Enhancing Human Preadipocyte Differentiation

The following example describes the optimization of methods used todifferentiate human preadipocytes. The example details a thoroughanalyses of known and potential agents and conditions for promotinghuman preadipocyte differentiation. Experiments were performed withabdominal subcutaneous as well as mesenteric and omental preadipocytes.A variety of conditions were tested using the fatty acid-basedcellulomic assay described in Example 4. Optimization of differentiationconditions enhances the usefulness of human preadipocytes in screeningfor modulators (e.g., inhibitors) of obesity. TABLE 1 Sample Dex (M)Uptake Ratio % Diff. Undiff.  0 1 0 Diff.  0 28 50 Diff. 10⁻¹⁰ 49 60Diff. 10⁻⁷ 79 70 Diff. 10⁻⁴ 5 30Variation of Dexamethasone Concentration to Optimize Differentiation

Table 1 shows effects of varying dexamethasone concentration on theratio of fatty acid uptake by cells exposed to differentiation mediumfor 10 d to that by undifferentiated cells (signal-to-noise) and extentof differentiation (% cells containing doubly-refractile dropletsevident by low power phase contrast microscopy). Briefly, humanpreadipocytes were cultured in either a control basal medium that doesnot promote differentiation (Undiff.) or a suboptimal differentiationmedium (Diff.), to which various concentrations of dexamethasone (Dex.)were added for 10 d. The fatty acid uptake ratio was expressed as afunction of fatty acid uptake in control medium. % Diff. represents theproportion of cells that developed doubly refractile lipid dropletsvisible by phase contrast microscopy by observers unaware of cultureconditions. Means of 4 studies are shown. CoV was also determined (notshown) to monitor assay reliability.

Increasing Time in Differentiation Medium Results in Increasing FattyAcid Uptake

The amount of time that preadipocytes were exposed to differentiationmedium was investigated. Table 2 depicts the results of experiments inwhich preadipocytes were exposed to differentiation medium for theamount of time shown and the fluorescence was measured. Day Fluor. AvgSt. Dev. 0 733 725 729 8 3 715 710 713 5 0 708 696 702 12 7 725 727 7262 0 690 675 683 15 11 654 662 658 8 0 696 723 710 27 15 684 705 695 21 0744 748 746 4 19 690 686 688 4

Both the average fluorescence and the standard deviation are reported.Measuring both the average fluorescence and the standard deviationfacilitates detection of signal, for example, in visceral preadipocyteswhich take longer to differentiate than abdominal subcutaneouspreadipocytes. Increasing differentiation times further increased thesignal-to-noise ratio in the fatty acid uptake assay.

Effects of Plating Density on Human Preadipocyte Differentiation

The effect of plating density was investigated. Plating density affectedextent of differentiation (Table 3) and was optimized. TABLE 3 PlatingMorphological Signal- Density Undifferentiated CoV Differentiated CoVDifferentiation to- (cells/cm × 10⁻⁴) (rfu: mean ± SEM) (%) (rfu: mean ±SEM) (%) (%) noise 1.5 753 ± 10 2.3 14166 ± 457 5.6 40 19 3.1 764 ± 143.2 16558 ± 831 8.7 60 22 4.7  771 ± 4.8 4.8 20249 ± 775 6.6 80 26 6.2751 ± 3  0.7 23140 ± 513 3.8 90 31 9.4 940 ± 61 11.2 23560 ± 299 2.2 9025

Increasing plating density was associated with an increase in thepercent of lipid-containing cells and improving reliability andsignal-to-noise ratios until densities of 6.2×10⁴ cells/cm² weresurpassed. Hence, this plating density was used in subsequent analyses.

Example 3 Methods to Isolate and Differentiate Human Preadipocytes

The following example details an optimized method of isolating anddifferentiating human preadipocytes. This method allows for high yieldof purified preadipocytes that have a high differentiative capacity.

-   -   1.) Prepare 1× Phosphate Buffered Saline/collagenase solution (3        mg collagenase/g of tissue and 1 ml 1× Phosphate Buffered        Saline/mg collagenase)+3.5% fatty acid free BSA and filter.    -   2.) Remove tissue from transport bottle with sterile forceps.        -   2A.) Omental tissue should be processed first since            digestion time is longer.            -   Omental tissue should be put into a sterile 100 mm dish                and sectioned into approx. 5 g pieces.            -   Each section of tissue should then be removed from the                dish with sterile forceps and transferred to a 50 ml                centrifuge tube that contains approx. 15 ml (3 ml/g of                tissue) of the 1× Phosphate Buffered Saline/collagenase                solution.        -   2B.) The above procedure should be repeated for subcutaneous            and mesenteric tissues.    -   3.) Mince the tissue in the solution to a fine consistency, be        sure to use sharp sterile scissors.    -   4.) Vortex each tube thoroughly and put the tubes in the water        bath to shake at 100 rpm and 37° C. Make sure the water bath is        full enough so that all tissue is submerged.    -   5.) The tubes should be vortexed thoroughly every 5-10 minutes.        The tubes should remain in the bath until all lumps appear        dissolved but not so long that a clear fat supernatant layer        appears.    -   6.) Once the solution appears homogeneous, each tube should be        vortexed and filtered through a sterile funnel containing a        double layered gauze filter. The samples should be filtered into        a sterile 50 ml centrifuge tube.    -   7.) Centrifuge at 1000 rpm for 10 min.    -   8.) Gently aspirate off fatty layer and most of the supernatant        leaving the pellet.    -   9.) Resuspend the pellet in 10 ml of Erythrocyte Lysing Buffer,        vortex, and shake in water bath at 37° C. and 100 rpm for 5 min.    -   10.) Remove from water bath, vortex, and centrifuge at 1000 rpm        for 10 min.    -   11.) Aspirate off most of the supernatant leaving the pellet.    -   12.) Resuspend the pellet in 10 ml of plating medium with 10%        NuSerum, vortex, and plate overnight in 100 mm dishes. Store in        incubator at 37° C. and 5% CO₂.    -   13.) After 24 hours, wash the cells three times with 10 ml of        PBS/EDTA.    -   14.) After aspirating off the final wash and add 1 ml of the        trypsin solution (5 ml of 10× trypsin+45 ml of PBS/EDTA) to the        100 mm dishes.    -   15.) Incubate the cells at 37° C. and 5% CO₂ until the majority        of the cells have lifted.    -   16.) To each dish add 5 ml of plating medium with 10% NuSerum to        inactivate the trypsin.    -   17.) Wash the cells from the dish with a narrow tip 5 ml        pipette. Transfer the liquid cell suspension to a 50 ml        centrifuge tube.    -   18.) Vortex and then centrifuge at 1000 rpm for 10 min.    -   19.) Aspirate off most of the supernatant and resuspend the        pellet in plating medium with 10% NuSerum (5-10 ml depending on        the size of the pellet).    -   20.) Transfer 100 μl from each tube to an epindorf tube. To each        epindorf tube add 100 μt of Trypan blue and vortex. Count the        cells using a hemocytometer, counting all four quadrants. # of        cells=(cell count/4)×2×(1×10⁴) x (# ml cells are suspended in).    -   21.) Plate the cells on desired plates at a density of 5×10⁴        cells/cm² in plating medium with 10% NuSerum.    -   25. ) Incubate in plating medium with 10% NuSerum at 37° C. and        5%CO₂ until confluence is reached, changing media every 48        hours.    -   26.) At confluence remove plating medium and add either        differentiating medium (to differentiate the cells), split the        cells to passage them, or freeze the cells.

For differentiation, cells are seeded in culture dishes, flasks orplates at 1.5-6×10⁴ cells/cm², preferably 3×10⁴ cells/cm². Uponadherence, cells are 100% confluent, and PM is changed every two to fourdays until differentiation is begun, two to 14 days after seeding.

Cells are differentiated for the first three days in freshly prepareddifferentiation medium (DM) consisting of DMEM/F12, HEPES, sodiumbicarbonate, penicillin, streptomycin, L-glutamine, transferrin, biotin,human insulin, panthothenic acid, fetuin, dexamethasone,triiodo-L-thyronine, rosiglitazone, and isobutylmethylxanthine (IBMX).Thereafter, IBMX may be reduced to 0-50% of the DM concentration.

On day 3 of differentiation, microscopic examination reveals that thecells have attained a rounded morphology. DM is changed on day 3 ofdifferentiation to DM without IBMX (DM2).

On day 6 of differentiation, the cells are more rounded in morphologyand have several tiny triglyceride droplets in their cytoplasm. DM ischanged on day 6 of differentiation to DM without IBMX, rosiglitazone,insulin or dexamethasone (DM3). DM3 is changed thereafter every threedays.

By day 9 of differentiation the triglyceride droplets are more numerousand larger in size. Further maintenance of cells in DM3 increases theirtriglyceride droplet size and number until the droplets eventuallycoalesce into one large droplet to displace the cell nucleus.

Solutions and Media TABLE 4 Transport Medium Ingredients Molarity 500 ml1 Liter Sigma# 1X PBS N/A 500 ml 1 L Gibco Gentamicin N/A 0.5 ml 1 LG-1397 Amphotericin N/A 1 ml 2 ml A-2942 Penicillin  0.1 mM 0.0295 g0.059 g PEN-NA Streptomycin 0.06 mM 0.05 g 0.10 g S-6501 L-glutamine   2mM 0.1461 g 0.2922 g G-5763*pH to 7.4 using either HCl or NaOH and filter sterilize.*The transport medium should be kept in either an amber bottle orwrapped in foil since the amphtericin is light sensitive and can becomecytotoxic.

TABLE 5 Human Plating Medium with 10% NuSerum Ingredients Molarity 500ml 1 Liter Sigma# DMEM/F-12 N/A 450 ml 900 ml Gibco Hepes   13 mM 1.78 g3.90 g H-7006 NaHCO₃   29 mM 1.22 g 1.26 g S233-3 Penicillin  0.1 mM0.0295 g 0.059 g P-3032 Streptomycin 0.06 mM 0.05 g 0.1 g S-6501L-glutamine   2 mM 0.1461 g 0.2922 G-8540 dexamethasone*  0.1 uM 5 ul ofstock 10 ul of stock D-4902 Heat inactivated N/A 50 ml 100 ml BD NuSerumBioSciences #33-5504*pH to 7.4 using either HCl or NaOH and filter sterilize.*dexamethasone stock: Use 4 mg per 1 ml of ethanol

TABLE 6 Erythrocyte Lysing Buffer Ingredients Molarity 300 ml 600 mlSigma# NH₄Cl 154 mM 2.47 g 4.94 g A-0171 KHCO₃ 10 mM 0.3 g 0.6 g P-7682EDTA <1 mM 0.011 g 0.022 g E-5134*pH to 7.4 using either HCl or NaOH and filter sterilize.

TABLE 7 α-MEM10 Medium Ingredient Concentration 1000 ml Catalog # waterN/A 900 ml N/A α-MEM N/A Gibco BRL sodium bicarbonate 26.8 mM 2.25 gFisher S233-3 penicillin  0.1 mM 59 mg PEN-NA streptomycin 0.06 mM 100mg S-6501 fetal bovine serum N/A 100 ml Gibco BRL- pH to 7.2 using hydrochloric acid and/or sodium hydroxide andfilter-sterilize through 0.2 um membrane.

TABLE 8 Cell Washing Buffer Ingredient Concentration 500 ml Catalog #water N/A 450 ml N/A 10X PBS N/A  50 ml Gibco BRL EDTA 0.7 mM mg E-5134- pH to 7.4 using hydrochloric acid and/or sodium hydroxide andfilter-sterilize through 0.2 um membrane.

TABLE 9 Trypsin Buffer Ingredient Concentration 500 ml Catalog #PBS/EDTA N/A 450 ml see Table 5 10X Trypsin 0.25X  50 ml Gibco BRL- pH to 7.4 using hydrochloric acid and/or sodium hydroxide andfilter-sterilize through 0.2 um membrane.

TABLE 10 Plating Medium (PM) Ingredient Concentration 1000 ml Catalog #water N/A 900 ml N/A DMEM/F12 N/A 12.0 g Gibco BRL HEPES,   15 mM 3.90 gH-7006 sodium salt sodium   15 mM 1.26 g Fisher S233-3 bicarbonatepenicillin  0.1 mM 59 mg PEN-NA streptomycin 0.06 mM 0.1 g S-6501L-glutamine   2 mM 0.2922 g G-5763 dexamethasone  0.1 μM 10 μl of stock*D-4902 NuSerum N/A 100 ml- pH to 7.2 using hydrochloric acid and/or sodium hydroxide andfilter-sterilize through 0.2 um membrane.*Dexamethasone stack is prepared fresh by dissolving 4 mg in 1 ml of100% ethanol; vortex until dissolved.

TABLE 11 Addition Volumes of PBS/EDTA and Trypsin Culture Dish/Flask mlPBS/EDTA ml Trypsin 100 mm dish 2 1.5  25 cm² T-flask 2 1  75 cm²T-flask 3 3 150 cm² T-flask 4 4 225 cm² T-flask 5 5

TABLE 12 Human Adipocyte Differentiation (HAD) Medium (DM) IngredientsMolarity 1 Liter Sigma # water N/A 900 ml N/A DMEM/F12 N/A 1 packageGibco Powder Penicillin 0.08 mM 0.0295 g PEN-NA Streptomycin 0.03 mM0.050 g S-6051 NAHCO₃ 25 mM 1.26 g Fisher S233-3 Hepes 23 mM 3.90 gH-7006 Transferin N/A 0.010 g T-6549 Biotin 0.03 mM 0.0081 B-4639L-glutamine 2 mM 0.2922 G-5736 Human Insulin 4 × 10⁻⁴ mM 0.00287 gI-2059 Pantothenic Acid 0.02 mM 0.00405 g P-5155 Fetuin N/A 1.0 g F-2379T3 (prepare 0.013 g in 10 ml; use 1 μl/L) T-6397 Dexamethasone (prepare0.004 g in 1 ml ethanol; D-4902 use 10 μl/L) BRL/rosiglitizone (prepare1 mg/280 μl DMSO; use 100 μl/L)*For DM + IBMX add 1.2 × 10−6 g/ml*pH before filtering to 7.4, use either HCl or NaOH

Plating medium (PM) may be changed on the third day post-seeding. Thenumber of days cells are maintained post-confluency prior todifferentiation may vary from 24 hours to 14 days, or beyond, with PMchanges occurring every two to three days. The seeding density ofpreadipocytes may range from 1.5-6×10⁴ cells/cm².

Example 4 Development of a Fatty Acid-Based Celluomic Assay for HumanPreadipocytes

This Example describes the development, optimization and validation of afatty acid-based celluomic assay for human preadipocytes. The assay isuseful for (1) detecting differences in the extent of adipogenesis ofhuman preadipocytes, in particular, in small numbers of humanpreadipocytes. Such an assay is particularly useful in performinghigh-throughput screens for fat-modulatory agents as well as foroptimizing conditions for culturing primary human adipocytes (see e.g.,Examples 1-3, above). The assay is equally useful for monitoring theextent of differentiation or de-differentiation of human adipocytes.

To establish a rapid, 96 well format, automated assay of fatty aciduptake, the reproducibility and signal-to-noise ratios of four differentfatty acid-fluorescent dye conjugates in undifferentiated anddifferentiated preadipocytes was determined. 3T3-L1 cells were used tocompare the fluorescent dyes, the best of which was then selected forthe human preadipocyte system. The fluorescent fatty acids tested were

1. 16-(9-anthroyloxy) palmitic acid (16-AP),

-   -   2. 12-(9-anthroyloxy) oleic acid (12-AO),    -   3. 4,4-difluoro-5-(2-thienyl)        1-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY        558/568 C12), and    -   4.        4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic        acid (BODIPY 500/510 C12).

These fatty acid-dye conjugates were chosen because their final size isclose to that of physiologically relevant C:18 fatty acids, the mostabundant in human fat cells and in the diet (Kokatnur, M., et al.(1979)Am. J. Clin. Nutr. 32:2198-205; Llado, I., et al. (1996) Biochem. Mol.Biol. Int. 40:295-303)). Furthermore, these dyes are vital, allowing thecells to be re-used in additional assays. 3T3-L1 cells were seeded inthe wells of a 96-well plate at a density of 3.3×10³ cells/cm² (1×10³cells/well). After reaching confluence, cells were exposed todifferentiation medium for 0 and 9 days. The wells were washed withassay buffer (150 mM NAZI, 10 mM NaPO₄; 3 mM KCl; 10 μM CaCl₂; 1 mMMgCl₂; 25 mM glucose; pH 7.4) containing 20 μM fatty acid-free BSA,pre-warmed to 37° C. The fluorescent fatty acids were added in assaybuffer to a final concentration of 10 μM, and incubated at 37° C. forone hour. Wells were washed with assay buffer containing 0.1% BSA.Results are shown in Table 13. TABLE 13 Comparison of fluorescent fattyacids for uptake by 3T3-L1 cells. Results represent means ± SEM RawFluorescent Units Fluor. Dye Day 0 Day 9 12-AO 3613 ± 160 10960 ± 117216-AP 2944 ± 404  8528 ± 20 BODIPY 500/510 C12  404 ± 21 56869 ± 2093BODIPY 558/568 C12  116 ± 9  3622 ± 641Day 0: Cells were not incubated in differentiation medium.Day 9: Cells were incubated 9 days in differentiation medium prior tothe lipid uptake assay.

BODIPY 500/510 C12 dye had the best signal-to-noise ratio of all thefluorescent fatty acids tested. Therefore, this dye was chosen forfurther investigation. In separate experiments, the dye was tested atdifferent time points for uptake by the 3T3-L1 cells. TABLE 14 BODIPYuptake increases with incubation time. 3T3-L1 cells were incubated with10 μM BODIPY 500/510 C12 for the times indicated. Time Day 0 Day 8 St.Dev.  10 min 1290.263 1198.325 1525.697  30 min 510.3125 18518.67118.4417 120 min 686.9375 46363 107.1977 240 min 1104.125 49153.33329.7533Means of triplicates ± SEM are shown.rfu: raw fluorescent units.

The results shown in Table 14 indicate that the minimum time requiredfor optimum assay results is between 30 and 120 minutes with 10 FMBODIPY 500/510 C 12. The dye was also tested for uptake at variousconcentrations, and 10 μM was the concentration that gave the bestresults. The data presented in Table 14 also demonstrate the excellentsignal-to-noise ratio (day 8 vs. day 0) for cells that were incubatedwith dye for 120 min. or more.

Table 15 indicates the results of experiments in which 3T3-L1 cells wereexposed to a differentiation inducing medium from day 0. Fatty aciduptake was assayed by exposing cells to BODPY for 120 minutes at each ofthe times represented. The results indicate that the longer apreadipocyte is exposed to differentiation medium (i.e., the moredifferentiated the cell is) the more dye uptake is observed. TABLE 15The Effect of Differentiation on Fatty Acid Uptake Day Fluor. Units St.Dev. 0 1166.67 196.869 2 5006.75 1144.76 4 10884.5 3932.8 6 15441.83011.79 8 18685 4491.85 10 31645.8 12338.2 12 37910 11694.7

The use of 3T3-L1 cells in the development of the lipid uptake assay ina high-throughput format facilitated the development of an assay usinghuman preadipocytes.

The BODIPY 500/510 C12 fatty acid uptake assay was optimized in thehuman preadipocyte system. The reliability of the assay was increasedsubstantially. When the methods developed in 3T3-L1 cells were firstused in human preadipocytes, the intra-assay standard coefficient ofvariation (CoV) was 40%. Once assay methods were optimized the CoV wasreduced to 3.8%. To achieve this improvement in reliability, a varietyof culture plates, dye incubation times, washing conditions,fluorimetric settings, plating densities, and times between induction ofdifferentiation and assay were tested. By increasing plating density to6.2×10⁴ cells/cm², the CoV was reduced, the extent of differentiationincreased, and the signal-to-noise ratio enhanced. By increasingincubation time, both reliability and signal-to-noise ratio were furtherincreased.

Example 5 Verification of Fatty Acid Uptake Assay as an Indicator ofAdipogenic Differentiation and Verification of Improved Human AdipocyteDifferentiation Methods

Abdominal subcutaneous preadipocytes were prepared as previouslydescribed and parallel, fourth-passage cultures were treated for 10 dayswith the Human Adipocyte Differentiation (HAD) medium of Table 12.Control cells were differentiated according to a published protocol orwere cultured in a medium that does not promote lipid accumulation. Thecells were assayed for fluorescent fatty acid assay uptake, as describedherein. Using the published differentiation method, even withisobutylmethylxanthine (IBMX) treatment from days 1 to 3 (which furtherimproves differentiation), only an 8 fold increase in uptake occurredcompared to undifferentiated control cells (CoV=18%; N=4). In parallelcultures treated using HAD medium, the increase was 129 fold (CoV=1%).These conditions were also tested in G3PD and aP2 secondary assays asfollows.

Gycerol-3-phosphate dehydrogenase (G3PD) activity assay. G3PD expressionincreases midway through adipogenesis and the G3PD promoter is activatedby both C/EBPα and PPARγ, reflecting activity of these key adipogenicpathways. G3PD activity was measured as described by Sottile and Seuwen(2001) Analytical Biochemistry 293:124-128. G3PD may be measured insupernatants of cell homogenates by following NADH disappearancespectrophotometrically. The assay is simple and can be done usingrelatively small numbers of cells. Preferred assay parameters are asfollows: CoV<1.6%; signal-to-noise 34-fold; minimal detectable limit:0.05 units (nmole dihydroxyacetone phosphate/ml×min); minimal detectabledifference: 0.019 units; activity in undifferentiated humanpreadipocytes: 5.4 units/10⁶ cells).

Western assay for aP2 and competimer rtPCR assay for PPARγ. Severaldifferent antibodies and primer sets were tested and it was decided touse Western assays for aP2 and competimer rtPCR for PPARγ for monitoringdifferentiation of preadipocytes. aP2 expression is very specific foradipose cells, undergoes a large increase during differentiation, and isfat depot-dependent. The aP2 Western antibody was sensitive and provideda linear response.

G3PD activity in human adipocytes cultured in the HAD medium of Table 10was 184.0 units. In adipocytes cultured according to the publishedmethod, activity was 9.3 units. In control undifferentiated cultures,activity was 5.4 units. aP2 protein was also more abundant in the cellsdifferentiated with HAD medium.

Thus, the differentiation methodology of the instant invention resultsin enhanced differentiation even when using subcultured (i.e.,fourth-passage) cells, thus greatly reducing costs and the number of fatsamples required. The differentiation methodology of the instantinvention also produces differentiated preadipocytes more rapidly thanpublished methods, saving time, medium changes, and associated expenseand contamination risk.

Example 6 HTS for Compounds that Inhibit FA* Accumulation in HumanSubcutaneous Adipocytes

Having optimized the fatty acid uptake assay using rodent and humanadipocytes and verified that the assay is a true indicator of phenotypicdifferentiation of adipocytes, high-throughput screening assays weredeveloped featuring human preadipocytes isolated and cultured accordingto the present invention.

Primary screens for inhibition of fatty acid uptake are carried outusing human subcutaneous preadipocytes in each of two assay formats. Ina first assay format, cells are exposed to natural product and syntheticlibrary components early in the differentiation process (days 1-3following addition of differentiation medium). In a second assay format,cells are exposed to library components late in the process (days 7-10following addition of differentiation medium), after at least 80% ofcells have differentiated.

Subcutaneous human preadipocytes (passage 3-5) are seeded in platingmedium at 3.0×10⁴ cells/cm² in 96-well or 384-well plates, giving 100%confluence upon adherence. Plating medium is exchanged 48- to 120-hourslater for differentiation medium plus IBMX to initiate adipocytedifferentiation. After 72 hours, differentiation medium containing alower concentration of IBMX is used. This medium is changed thereafterevery three to four days until cells are assayed for fluorescent fattyacid (FA*) accumulation.

Cells are treated in triplicate with compounds at variousconcentrations. Compounds are first diluted in a phosphate-bufferedsaline solution containing 0.1% fatty-acid free bovine serum albumin—andthen added to cells. Negative control cells are treated with DMSO, thesolvent used for initially dissolving compounds, at 0.1% finalconcentration. Positive control cells are treated with carbonyl cyanidep-(trifluoromethoxy)-phenylhydrazone (FCCP, a potent uncoupler ofoxidative phosphorylation in mitochondria). Cells are then allowed todifferentiate for 72 hours more before being assayed for FA*accumulation as follows.

Differentiated adipocytes are pre-washed three times with a fatty acidbuffer (FAB) containing: DMEM/F12 (1:1), 15 mM HEPES, 15 mM NaHCO3, and2 mM glutamine. Cells are then incubated with FA* at 10 μM in FAB plus0. 1% fatty-acid free BSA (FAB+) for 4 hours at 37° C. Cells are thenpost-washed five times with FAB+ and FA* accumulation is measured on amicroplate reader at excitation/emission 485/635 nm.% efficacy of compound on inhibiting FA* accumulation is calculated asfollows:% Efficacy=100−(sample fluorescence/negative control fluorescence×100).

Subsequent determination of compound toxicity is measured by incubatingcells with one tenth of total cell volume of Alamar Blue (an indicatorof cellular metabolism) for up to four hours before measuringfluorescence of the reduced compound. Cell survival is measured bymonitoring the fluorometric change produced in the dye upon itsreduction by living cells (Fields, R. et al. (1993) Am. Biotechnol. Lab.11:48-50).

Toxicity is calculated as follows:% Toxicity=100−(sample fluorescence/negative control fluorescence×100).

Table 14 depicts the results of screening various concentrations of FCCPfor both inhibition of FA* accumulation and cellular toxicity indifferentiating adipocytes. TABLE 14 FCCP: 0 μM 0.3 μM 1 μM 3 μM %Efficacy: 0 42 79 90 % Toxicity: 0 −1 −4 5

In a first round of screening, 50,000 combinatorial compounds and 10,000natural product compounds obtained from microbial extracts were screenedfor efficacy. Primary hits were selected as those producing a greaterthan 85% decrease in fatty acid content as compared to controls. Thisscreening is described in detail in U.S. application Ser. No.10/201,588, which is incorporated herein by reference. Primary hits wereevaluated further to determine concentration response relationships forefficacy and toxicity. Natural compound hits resulted in a greater than80% decrease in fatty acid content without any observable toxicity tohuman cells (adipocytes and fibroblasts). Combinatorial compound hitsshow comparable efficacy at low nanomolar levels without toxicity. Usingthese assays, multiple, non-toxic, chemically dissimilar hits have beenidentified.

Hits are further characterized using secondary screens to evaluate thebiochemical mechanism of action and identify molecular targets orpathways effected by the hit. Compounds affecting accumulation of fattyacid may act via a variety of relevant mechanisms such as decreasingfatty acid uptake and/or triglyceride synthesis/storage (lipogenesis),or increasing triglyceride breakdown (lipolysis) and/or fatty acidoxidation. In addition, hits are evaluated for their effects on thedifferentiation process and, optionally, are evaluated for theirspecificity toward adipocytes from the distinct anatomical depots (seeExample 10).

Example 7 Identification of Agent for Use in Fatty AcidAccumulation/Uptake Modulation

Preadipocytes were isolated from human subcutaneous fat tissue andcultured as described previously. Cells were seeded in a 384-well plateat 3×10⁴ cells/cm² (giving 100% confluency upon adherence) andmaintained at 100% confluency for five days in 50 μl of Plating Medium(PM), then differentiated.

Day 0 of Differentiation: Plating medium was completely exchanged with50 μl freshly prepared Differentiation Medium (DM), described in Example3 including 540 micromolar IBMX.

Day 3 of Differentiation: 40 μl of the medium ( 4/5 of total volume) wasexchanged with DM minus IBMX (DM2).

Day 6 of Differentiation: All 50 I¹ of DM2 was exchanged with DM minusIBMX, rosiglitazone, insulin and dexamethasone (DM3). A test agent wasadded to cells at various concentrations (1000, 300, 100, 30 and 10 nM).

Day 9 of Differentiation: To remove unincorporated test agent, the cellplate was pre-washed three times in Fatty Acid Buffer containing 0. 1%fatty acid-free BSA (FAB). Fluorescent fatty acid (FA*, D-3823,Molecular Probes, Eugene Oreg., 10 mM stock solution in DMSO, stored at-20° C.) was diluted in FAB, then added to cells at 5 μM concentration.

Cells were incubated for four hours at 5% carbon dioxide/37° C., afterwhich the plate was post-washed three times in a manner similar to thepre-wash.

Fluorescence of FA* incorporated into adipocytes was determined with amicroplate reader. The % efficacy of the test agent on inhibiting FA*uptake and/or accumulation was determined as described previously.

To determine compound toxicity toward cells, the plate then wasincubated for three hours more at 5% carbon dioxide/37° C. after 5 μl (1/10) addition of Alamar Blue (BioSource International, Inc.) to thesample wells. Fluorescence of reduced Alamar Blue was determined with amicroplate reader. The % toxicity of the test agent was determined asdescribed previously.

Adipogenesis

The test agent was assayed for its effect on expression of G3PD, anindicator of the extent of adipogenesis in differentiated preadipocytes.G3PD may be measured by monitoring NADH disappearancespectrophotometrically as previously described. Preadipocytes wereisolated from human subcutaneous fat tissue and cultured as describedpreviously. Cells were seeded in a 96-well plate at 3×10⁴ cells/cm² andmaintained at 100% confluency for five days in 200 μl of Plating Medium(PM). Preadipocytes were differentiated as described above in thepresence of the agent, except that volume changes were 200 μl on Day 0of Differentiation and 160 μL on Day 3 of Differentiation.

Day 6 of Differentiation: All 200 μl of DM was exchanged with DM minusIBMX, rosiglitazone, insulin and dexamethasone. The test agent was addedto cells at 30 μM final concentration (0.1% DMSO). 1 nM of human tumornecrosis factor alpha (TNFα, a cytokine that inhibits preadipocytedifferentiation and adipogenesis) also was added to cells in a similarmanner as a positive control. 0.1% DMSO also was added to cells as anegative control.

Day 9 of Differentiation: Treated cells were pre-washed three times withFatty Acid Buffer (FAB, described in previous section) with 160 μlvolume exchanges to remove the DM and compound. Cells were incubated forfour hours at 5% carbon dioxide/37° C. (under conditions describedabove), then assayed for G3PD activity.

Lipogenesis

The test agent was assayed for its ability to modulate triglyceridesynthesis in adipocytes, as monitored by 14-carbon labeled glucoseconversion to the glycerol backbone of triglycerides. Preadipocytes wereisolated from human subcutaneous fat tissue and cultured as describedpreviously. Cells were seeded in a 12-well plate at 3×10⁴ cells/cm² andmaintained at 100% confluency for five days in 2 ml of PM. Preadipocyteswere differentiated as described above, except that volume changes were2 ml on Day 0 Differentiation and 1.6 ml on Day 3 Differentiation.

Day 6 Differentiation: All 2 ml of DM was exchanged with DM minus IBMX,rosiglitazone, insulin and dexamethasone. The test agent was added tocells at 30 μM final concentration (0.1% DMSO). 10 μM wortmannin (aphosphoinositide-3 kinase inhibitor that affects glucose metabolism incells) also was added to cells in a similar manner as a positivecontrol. 0.1% DMSO also was added to cells as a negative control. Day 9Differentiation: Treated cells were pre-washed once with 2 ml ofLipogenesis Buffer (described below), pre-warmed to 37° C. This volumewas then exchanged with 2 ml of Lipogenesis Buffer plus 10 nM insulinand.10 μM uniformly labeled 14-carbon glucose. The cell plate wasincubated for four hours at 5% carbon dioxide/37° C., then the treatedcell wells were post-washed twice with Lipogenesis Buffer, then placedon ice. Ingredient Concentration 1000 ml Catalog # water N/A 900 ml N/ADMEM* N/A 12.0 g Gibco BRL HEPES, sodium salt 15 mM 3.90 g H-7006 sodiumbicarbonate 15 mM 1.26 g Fisher S233-3 L-glutamine  2 mM 0.2922 g G-5763glucose  5 mM 0.900 g fatty acid-free BSA 0.1% 1.0 g Intergen 3320-01pH to 7.2 using hydrochloric acid and/or sodium hydroxide andfilter-sterilize through 0.2 um membrane.*DMEM minus glucose

Lipid Extraction: All of Lipogenesis Buffer was removed from the treatedcell well, and 200 ul of ice-cold 1× trypsin solution was added. After afew minutes of incubation, cells were detached from the well with a cellscraper and transferred to a glass test tube on ice. 175 ×l of ice-cold21.4 mM hydrochloric acid were added to the well and pipeted up and downseveral times to remove any residual cells attached; the contents weretransferred to the same test tube on ice. 1.5 ml of chloroform:methanol(2:1) was added to the test tube and vortexed to extract lipids into theorganic phase. The test tube was centrifuged at 400×g for five minutesat room temperature to separate the organic and aqueous phases. Thelower organic layer was removed with a glass pipet and transferred to anew screwtop glass test tube. The contents were dried down undernitrogen gas and stored at −20° C. until thin layer chromatography (TLC)separation.

TLC: The lipid film was redissolved in 50 μl of chloroform, and 5 μl ofthe solution were transferred to a scintillation vial for 14-carbonquantitation. Another 5 μl were spotted on Baker Silica Gel TLC plates(plastic backing), and then run in a chamber with chloroform:diethylether:acetic acid. The lipid spots were visualized with gaseous iodine,and the triglyceride spot was cut out and placed in a scintillation vialfor 14-carbon quantitation.

Lipolysis

The test agent was assayed for its ability to modulate lipolysis inadipocytes, as monitored by release of pre-loaded fluorescent fatty acid(FA*) from triglyceride droplets in the cells. Preadipocytes wereisolated from human subcutaneous fat tissue and cultured as describedpreviously. Cells were seeded in a 384-well plate at 3×10⁴ cells/cm² andmaintained at 100% confluency for five days in 50 ul of PM.Preadipocytes were differentiated as described above.

Day 8 Differentiation: FA* (D-3823, Molecular Probes, Eugene Oreg.) wasdiluted in FAB and added to cells at 10 μM.

Day 9 Differentiation: Treated cells were pre-washed three times withFAB to remove unincorporated FA*. Fluorescence of loaded FA* in thetriglyceride droplets of cells was measured with a microplate reader Thetest agent was added to a portion of the cells (1000, 300, 100, 30 and10 nM). Forskolin (an inhibitor of adenylate cyclase) was added to cellsin the micromolar range in a similar manner as a positive control. 0.1%DMSO was added to cells as a negative control. Cells were incubated forfour hours at 5% carbon dioxide/37° C. Cells then were post-washed threetimes with FAB to remove released FA*, and fluorescence was measured.The ratio of FA* fluorescence before and after compound exposure wasdetermined to quantitate the extent of lipolysis.

The test agent was shown to inhibit lipolysis.

Oxidation

The test agent was assayed for its ability to modulate oxygenconsumption in adipocytes, as monitored by quantitation of oxygen levelsin the medium surrounding cells using an oxygen binding fluorescentprobe in a 96-well round bottom plate (BD BioScience Oxygen BiosensorSystem (OBS)). Preadipocytes were isolated from human subcutaneous fattissue and cultured as described previously. Cells were seeded in a6-well plate at 3×10⁴ cells/cm² and maintained at 100% confluency forfive days in 3 ml of PM. Preadipocytes were differentiated as described,except that volume changes were 3 ml on Day 0 Differentiation and 2.4 mlon Day 3 Differentiation.

Day 6 Differentiation: All 3 ml of DM were-exchanged with DM minus IBMX,rosiglitazone, insulin and dexamethasone. The test compound was added tocells at 30 μM final concentration (0.1% DMSO). 10 ,μM carbonyl cyanidep-(trifluoromethoxy)-phenylhydrazone (FCCP, a potent uncoupler ofoxidative phosphorylation in mitochondria) also was added to cells in asimilar manner as a positive control. 0.1% DMSO also was added to cellsas a negative control.

Day 9 Differentiation: Since the 96-well OBS plate has theoxygen-sensitive fluorescent probe attached in a silica matrix to theround-bottom well, adherent cells cannot attach and grow well.Therefore, to use this system the differentiated preadipocytes must bedetached from the 6-well plate and placed in the OBS plate insuspension.

Treated cells in the 6-well plate were pre-washed once with 2 ml ofPBS/EDTA (see previous section for recipe), then incubated with 250 μlof 1× trypsin in PBS/EDTA for five minutes, with swirling every 30seconds. 500 μl of PM minus penicillin and streptomycin, and also minusphenol red (MediaTech 90-090-PC; the phenol red color can interfere withthe OBS probe fluorescence) was added, and the cells were gentlydetached with pipetting. More PM was added such that transferring 200 μlinto an OBS well gave 25,000 cells/well.

Cells in the 6-well plate not treated with compounds also were detachedand placed in OBS wells in a similar manner. These cells then weretreated with the test agent (30 μM), FCCP (1 μM) or DMSO (0.1%) foracute exposure, to compare to cells treated for three days. Empty OBSwells also were treated with water or 100 mM sodium sulfite todemonstrate the full range of probe fluorescence (sodium sulfite willremove all oxygen from the system to allow complete probe fluorescence).

After the addition of cells, compounds and sodium sulfite, the OBS platefluorescence was determined with a microplate reader. After incubationfor four hours at 5% carbon dioxide/37° C., the plate was read again.The change in fluorescence was used to quantitate the amount of oxygenconsumption in the medium surrounding the cells.

Example 8 Animal Studies

The agent from Example 7 was tested for its ability to inhibit weightgain in Sprauge-Dawley rats fed a commercial high fat diet (HarlanTeklad, Product No. TD 98211). Weight gain was significantly less inrats injected with the agent as compared to controls.

Example 9 Effects of Fat Depot Origin on Human Preadipocyte Replication,Differentiation, and Fatty Acid Handling

The following example describes the effects of fat depot origin onpreadipocyte replication, differentiation and fatty acid handling. Theexperiments show that preadipocytes from the three different fat depotsrespond differently to differentiation as evident through differentialexpression of differentiation markers. The example also providesmethodology that allows for detection of substantial differences incharacteristics of preadipocytes from different regions.

Using rat preadipocytes, it was determined that regional differences inexpression of fatty acid binding proteins (aP2 and keratinocyte lipidbinding protein), as well as enzymes of fatty acid metabolism (includingcarnitine palmitoyl transferase I and G3PD) contribute to interdepotvariation in fatty acid uptake and esterification. Regional differencesin cultured preadipocyte aP2 levels were reflected in regionaldifferences in fat cell aP2 expression iii vivo. Interdepot differenceswere found in human preadipocyte aP2 expression and G3PD activity aswell. These likely contribute to interdepot differences in fatty acidhandling and are related to variation in capacity for differentiation.aP2 was highest in differentiating human abdominal subcutaneouspreadipocytes, lower in mesenteric preadipocytes, and lowest in omentalpreadipocytes as shown by a western blot using a aP2 specific antibodyafter 10 days. Even in omental preadipocytes, aP2 expression increasedwith differentiation, particularly in primary culture.

After 10 days of treatment with differentiation medium, abdominalsubcutaneous preadipocyte G3PD activity was 235.2 units/million cells,mesenteric was 28.2, and omental 17.2, while undifferentiated controlcell G3PD was 5.4. Others have also found interdepot variation in humanpreadipocyte G3PD activity (Hauner, H., et al. (1991) Int. J. Obesity15:121-6)). After longer periods of differentiation medium exposure,G3PD activity increased. Hence, even after relatively short periods ofexposure to differentiation inducing media, distinct patterns of fattyacid binding protein expression and lipogenic enzyme activities occur inpreadipocytes cultured from different regions of the same individual,indicating the feasibility of developing treatments that havedifferential effects on fat tissue from different regions.

Others have reported the PPARγ expression does not differ among humansubcutaneous and omental preadipocytes even though they did find thatthiazolidinediones, which are PPARγ activating ligands, caused moreextensive differentiation of subcutaneous than omental cells (Adams, M.,et al. (1997) J. Clin. Invest. 100:3149-53)). By contrast, subcutaneous,omental, and mesenteric preadipocytes differentiated according to themethodology described herein exhibited significant differences in bothPPARγ2 mRNA and protein abundance. Moreover, interdepot differences inhuman preadipocyte cytokine release (previously reported in vivo) havebeen found. Particularly in the case of TNFα, these differences inpreadipocyte autocrine factor release may have important effects onregional patterns of fat tissue function. TNFα, for example, affectsdifferentiation and lipid accumulation, as well as activities of keyadipogenic transcription factors including PPARγ and C/EBPα (Ron, D., etal. (1992) J. Clin. Invest. 89:223-33; Stephens, J. M., et al (1992) J.Biol. Chem. 267:135804; Williams, P. M., et al. (1992) Mol. Endocrinol.6:1135-41; Zhang, B., et al. (1996) Mol. Endocrinol. 10: 1457-66;Szalkowski, D., et al. (1995) Endocrinology 135:1474-81)). Thus,multiple regulatory pathways important in controlling lipid accumulationdiffer among fat depots, and these differences in tissue characteristicsare reflected in preadipocytes cultured according to the instantmethodologies.

Example 10 Fat Depot-Specific Screening Assays

The following example provides methods to identify compounds (e.g.,small molecules, peptides, or peptidomimetics) that are capable ofmodulating the proliferation and/or growth of human adipocytes. Primaryscreens identify compounds that are capable of combating obesity byreducing visceral fat. Those compounds are then tested with omental andmesenteric cells to determine their ability to modulate preadipocytesfrom these depots.

Subcutaneous preadipocytes, which are more easily obtained in thequantities necessary, are used for the primary screen. Because thereduction of visceral fat is important for a drug designed to combatobesity, omental and mesenteric preadipocytes are used in secondaryscreens. The secondary screens may also identify compounds that have agreater activity on omental or mesenteric than on subcutaneouspreadipocytes. This information is important in the prioritization ofcompounds for further study.

The accumulation of visceral fat is more strongly associated withobesity-related diseases, such as diabetes, than is the accumulation offat in other depots. Therefore, potential drugs aimed at decreasingobesity should at the very least target all fat depots and, at best,target preferentially visceral fat. The primary screens for inhibitionof fatty acid uptake are carried out as described in Example 8 in eitherof the two assay formats. The secondary screens for inhibition of fattyacid uptake are similar to the primary screens, but utilize omental andmesenteric preadipocytes. Therefore, omental and mesenteric cells areexposed to hits from the primary screens using the two assay formatinvolving early and late addition of library components.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for identifying a fat modulatory compound, comprising: (a)obtaining a high-yield, essentially pure human preadipocyte population;(b) culturing said preadipocyte population under conditions sufficientto induce differentiation; (c) contacting said population with a testcompound at least 1-3 days following initial culture in thedifferentiation medium; (d) maintaining said population in the presenceof said test compound; and (e) assaying the population for fatty-aciduptake or accumulation, a detectable fluctuation in which indicates thatthe test compound is a fat modulatory compound.
 2. A method foridentifying a fat modulatory compound, comprising: (a) obtaining ahigh-yield, essentially pure human preadipocyte population; (b)culturing said preadipocyte population under conditions sufficient toinduce differentiation; (c) contacting said population with a testcompound at least 6-10 days following initial culture in thedifferentiation medium; (d) maintaining said population in the presenceof said test compound; and (e) assaying the population for fatty-aciduptake or accumulation, a detectable fluctuation in which indicates thatthe test compound is a fat modulatory compound.
 3. A method foridentifying a potential weight loss or anti-obesity agent, comprising:(a) obtaining a high-yield, essentially pure human preadipocytepopulation; (b) culturing said preadipocyte population under conditionssufficient to induce differentiation; (c) contacting said populationwith a test compound at least 1-3 days following initial culture in thedifferentiation medium; (d) maintaining said population in the presenceof said test compound; and (e) assaying the population for fatty-aciduptake or accumulation, a detectable inhibition of which identifies thetest compound as a potential weight loss or anti-obesity agent.
 4. Amethod for identifying a potential weight loss or anti-obesity agent,comprising: (a) obtaining a high-yield, essentially pure humanpreadipocyte population; (b) culturing said preadipocyte populationunder conditions sufficient to induce differentiation; (c) contactingsaid population with a test compound at least 6-10 days followinginitial culture in the differentiation medium; (d) maintaining saidpopulation in the presence of said test compound; and (e) assaying thepopulation for fatty-acid uptake or accumulation, a detectableinhibition of which identifies the test compound as a potential weightloss or anti-obesity agent.
 5. The method of claim 1, further comprisingthe step of determining the effect of the test compound on at least oneof adipogenesis, lipogenesis, lipolysis, and oxidation.
 6. The method ofclaim 3, further comprising the step of determining the effect of thetest compound on at least one of lipogenesis, lipolysis, and oxidationin the presence of said compound,wherein the effect is inhibition oflipogenesis, increase in lipolysis or increase in oxidation.
 7. Themethod of claim 1, wherein the conditions sufficient to inducedifferentiation comprise culturing said population in a serum-freedifferentiation medium.
 8. The method of claim 1, further comprising thesteps of determining the toxicity of said test compound on said humanpreadipocyte population.
 9. The method of claim 1 further comprising thestep of determining the toxicity of said test compound on a control cellpopulation.
 10. A method of identifying a potential weight loss oranti-obesity agent comprising: (a) selecting a fat modulatory compoundidentified according to claim 1, said compound being capable ofinhibiting fatty-acid uptake or accumulation in the absence ofsubstantial toxicity to said adipocyte population; and (b) determiningthe effect of the test compound on at least one of lipogenesis,lipolysis, and oxidation in the presence of said compound, wherein theeffect is inhibition of lipogenesis, increase in lipolysis or increasein oxidation identifies said compound as a potential weight loss oranti-obesity agent.
 11. The method of claim 10, further comprising thestep of determining the effect of the test compound on adipogenesis. 12.A method for identifying a target for an agent modulating weight gaincomprising: identifying a compound that inhibits differentiation ofpreadipocytes to adipocytes in the absence of substantial toxicity tosaid preadipocytes; and determining the presence or absence of analteration in lipogenesis, lipolysis, and oxidation in the presence ofsaid compound, wherein an alteration signifies said target.
 13. Themethod of claim 11 wherein adipogenesis or differentiation ofpreadipocytes to adipocytes is determined by assaying for G3PD activityor PPAR γ activity.
 14. The method of claim 11 wherein adipogenesis ordifferentiation of preadipocytes to adipocytes is determined by assayingfor expression of a gene selected from the group consisting of PPARγ,C/EBPα, C/EBPβ, aP2, and GLUT4.
 15. The method of claim 10 whereinlipogenesis is determined by measuring glucose to triglycerideconversion.
 16. The method of claim 10 wherein lipogenesis is determinedby measuring oleate to triglyceride conversion.
 17. The method of claim10 wherein lipolysis is determined by measuring release of labeled fattyacids from adipocytes.
 18. The method of claim 10 wherein lipolysis isdetermined by measuring glycerol release from adipocytes.
 19. The methodof claim 10 wherein lipolysis is determined by measuring increasedacrylodan-labeled intestinal fatty acid binding protein (ADIFAB)binding.
 20. The method of claim 10 wherein oxidation is determined bymeasuring oxygen consumption.
 21. A method for identifying a fatdepot-specific inhibitor of human preadipocyte differentiation,comprising: (a) identifying an inhibitor of human preadipocytedifferentiation in a preadipocyte population derived from a first depot;and (b) comparing the efficacy of said inhibitor in a preadipocytepopulation from a second depot, such that a fat depot-specific inhibitorof human preadipocyte differentiation is identified.
 22. The method ofclaim 21, wherein the first depot is subcutaneous fat depot and thesecond depot is an omental or mesenteric fat depot.
 23. A cellpopulation comprising human preadipocytes, wherein said population isessentially pure.
 24. The cell population of claim 23, wherein saidpopulation is at least 95% pure.
 25. The cell population of claim 23,wherein said population is at least 98% pure.
 26. The cell population ofclaim 23, wherein said population is at least 99% pure.
 27. The cellpopulation of claim 23, wherein the human preadipocytes are insuspension.
 28. The cell population of claim 23, wherein the humanpreadipocytes are adhered to a cell culture surface.
 29. The cellpopulation of claim 23, wherein the human preadipocytes are ofsubcutaneous origin.
 30. The cell population of claim 23, wherein thehuman preadipocytes are of mesenteric origin.
 31. The cell population ofclaim 23, wherein the human preadipocytes are of omental origin.
 32. Ahuman preadipocyte cell strain which maintains differentiative capacityover at least 8 passages.
 33. The cell strain of claim 32 whichmaintains differentiative capacity over at least 15 passages.
 34. Ahuman preadipocyte cell strain which maintains differentiative capacityover at least 25 passages.
 35. A high-yield process for obtaining ahuman preadipocyte cell population which is essentially pure,comprising: (a) isolating a mixed cell population from said fat tissuesample under conditions favoring a high preadipocyte yield; and (b)removing contaminants from said mixed cell population, such that theessentially pure human preadipocyte cell population is obtained.
 36. Theprocess of claim 35, wherein the conditions favoring a high preadipocyteyield comprise: (a) isolation within 0 to 24 hours following harvestingof said fat tissue; and (b) removal of undigested connective tissue fromthe mixed cell population utilizing gauze filters.
 37. The process ofclaim 35, wherein removing contaminants from said mixed cell populationcomprises: (a) removing contaminating erythrocytes; (b) removingcontaminating adherent cells; and (c) removing tissue and cellulardebris.
 38. The process of claim 37, wherein removing contaminatingerythrocytes is accomplished by incubating the mixed cell population inan erythrocyte lysis buffer.
 39. The process of claim 37, whereinremoving contaminating adherent cells is accomplished by adhering themixed cell population to a cell culture surface and preferentiallytrypsinizing the preadipocytes.
 40. The process of claim 35, whereinsaid fat tissue is of subcutaneous origin.
 41. The process of claim 35,wherein said fat tissue is of mesenteric origin.
 42. The process ofclaim 40, wherein the conditions favoring a high preadipocyte yieldfurther include coarsely mincing said fat tissue
 43. The process ofclaim 35, wherein said fat tissue is of omental origin.
 44. The processof claim 43, wherein the conditions favoring a high preadipocyte yieldfurther include: (a) finely mincing said fat tissue; and (b) digestingsaid fat tissue in the presence of a semi-artificial serum supplement orbovine serum albumin.
 45. The process of claim 35, wherein theessentially pure human preadipocyte cell population obtained is at least85% pure.
 46. The process of claim 35, wherein the essentially purehuman preadipocyte cell population obtained is at least 90% pure. 47.The process of claim 35, wherein the essentially pure human preadipocytecell population obtained is at least 95% pure.
 48. The process of claim35, wherein the yield is at least 10⁶ preadipocytes from 25 g of humanfat tissue
 49. The process of claim 35, wherein the yield is at least10⁶ preadipocytes from 10 g of human fat tissue
 50. The process of claim35, wherein the yield is at least 10⁶ preadipocytes from 5 g of humanfat tissue
 51. The process of claim 35, wherein the yield is at least10⁶ preadipocytes from 1 g of human fat tissue
 52. A method of obtaininga highly-differentiated human adipocyte cell culture, comprising: (a)obtaining a high-yield, essentially pure population of humanpreadipocytes; (b) plating said preadipocytes at a density sufficient toensure essentially 100% confluence upon adherence to the cell culturedish or plate; (c) maintaining said preadipocytes in a serum-freedifferentiation medium such that a highly-differentiated human adipocytecell culture is obtained.
 53. The method of claim 52, wherein the cellsare plated at a density of 3×10⁴ to 5×10⁴ cells per cm².
 54. The methodof claim 52, wherein the serum-free differentiation medium comprises abuffering component, glutamine, biotin, insulin, pantothenate,dexamethasone, triiodothyronine, rosiglitazone, fetuin, transferin, andisobutylmethylxanthine.
 55. A method of obtaining a high-yield,essentially pure culture of human preadipocytes comprising; (a)enzymatically dissociating a mixed-cell population from human fat tissuein serum-free, bovine serum (BSA)-containing isolation media; (b)isolating the mixed-cell population from non-cell contaminants usinggauze filters; (c) treating the mixed-cell population with anerythrocyte lysis buffer prior to plating the cell population; and (d)selectively trypsinizing the cell population and replating in aserum-free differentiation medium at a density sufficient to insureconfluence.
 56. A method of obtaining a highly-differentiated humanadipocyte culture comprising obtaining a high-yield, essentially pureculture of human preadipocytes according to the method of claim 55 andfurther maintaining the culture in the serum-free differentiation mediumsuch that a highly-differentiated human adipocyte cell culture isobtained.